Methods for treatment of nephrotic syndrome and related conditions

ABSTRACT

The present disclosure provides a polypeptide and method for treating and/or preventing nephrotic syndrome, such as but not limited to those associated with minimal change disease and membranous nephropathy, and conditions related to nephrotic syndrome, such as but not limited to, proteinuria and edema, as well as diabetic nephropathy, diabetes mellitus, lupus nephritis or primary glomerular disease. The present disclosure further provides methods for reducing proteinuria and other disease states as discussed herein. Such methods comprise the therapeutic delivery of an Angptl4 polypeptide or Angptl4 polypeptide derivative to a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/855,060, filed Sep. 15, 2015 (pending) U.S. application Ser. No.14/855,060 is a divisional of U.S. application Ser. No. 13/841,240,filed Mar. 15, 2013, now U.S. Pat. No. 9,139,629 (issued Sep. 22, 2015).U.S. application Ser. No. 13/841,240 is a continuation-in-part of U.S.application Ser. No. 13/364,962, filed on Feb. 2, 2012, now U.S. Pat.No. 9,475,850 (issued Oct. 25, 2016). U.S. application Ser. No.13/364,962 is a continuation of International ApplicationPCT/US11/39255, filed on Jun. 6, 2011 (expired). InternationalApplication PCT/US11/39255 cites for priority U.S. Application61/351,866, filed Jun. 5, 2010. U.S. application Ser. No. 13/364,962cites for priority U.S. Application 61/438,854, filed on Feb. 2, 2011.U.S. application Ser. No. 13/841,240 is incorporated herein by referencein its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersR01DK077073 and R01DK090035 awarded by the National Institute of Health.The government has certain rights in the invention.

FIELD OF THE DISCLOSURE

The present disclosure is directed to methods for the treatment andprevention of nephrotic syndrome and conditions related thereto, suchas, but not limited to, proteinuria and edema.

BACKGROUND

Nephrotic syndrome (NS) is a general term that refers to the loss ofprotein in the urine (proteinuria), hyperlipidemia (hypercholesterolemiaand hypertriglyceridemia), and edema. Nephrotic syndrome involveschanges in the pathology of cells in the kidney, such as podocytes.Proteinuria is defined as the presence of an excess of serum proteins inthe urine. Albuminuria, a specific type of proteinuria, is apathological condition wherein albumin is present in the urine.

Podocytes (or visceral epithelial cells) are cells in the outer layer ofthe glomerular capillary loop in the kidneys. The glomerulus filtersblood, holding back large molecules such as proteins, and passingthrough small molecules such as water, salts, and sugar, as the firststep in forming urine. The long projections, or “foot processes,” of thepodocytes wrap around the capillaries, and come to rest on theglomerular basement membrane. The foot processes are connected by aporous structure called the slit diaphragm. The innermost layer of theglomerular capillary loop is made of fenestrated endothelial cells.Kidneys affected by nephrotic syndrome have abnormalities in theglomerular capillary loop that cause leakage of blood proteins,resulting in proteinuria.

When protein is lost in the urine, its plasma concentration decreases,allowing water to move into other areas of the body, which leads toswelling known as edema. Edema is commonly observed in the feet andlegs, in the belly or abdomen (ascites), and around the eyes, but canoccur anywhere, especially in response to gravity. Additionally, becauseof this extra fluid that stays in the body, people often gain weight,experience fatigue and may find that they urinate less often

Many conditions are categorized as nephrotic syndromes, includingminimal change disease (MCD), focal segmental glomerulosclerosis (FSGS),membranous nephropathy (MN) (also called membranous glomerulonephritis,MGN), and membranoproliferative glomerulonephritis (MPGN). For yearspathologists found no changes in MCD tissue when viewing specimens underlight microscopy, hence the name minimal change disease. With the adventof electron microscopy, the changes now known as the hallmarks for thedisease include diffuse loss of podocyte foot processes, vacuolation ofthe podocyte foot processes, and growth of microvilli on the visceralepithelial cells. Diabetic nephropathy is the most common cause ofnephrotic syndrome.

Hypertriglyceridemia may occur due to changes in the activity of enzymesthat degrade triglycerides, such as lipoprotein lipase (LPL) (2-4).Certain proteins involved in the etiology of nephrotic syndrome andproteinuria, such angiopoietin-like 4 (Angptl4), inhibit the activity ofLPL.

The molecular basis of nephrotic syndrome is not known. Increased levelsof Angptl4 have been noted in nephrotic syndrome, such as MCD, MN/MGN,and MPGN, but increased circulating levels of Angptl4 have not beenassociated with causation of proteinuria in nephrotic syndrome. However,the role of Angptl4 in nephrotic syndrome, such as but not limited to,MCD, FSGS, MN/MGN, and MPGN, and related conditions, such as, but notlimited to, proteinuria have not been previously reported. Furthermore,the association of proteinuria and glucocorticoid sensitivity innephrotic syndrome and the link between proteinuria andhypertriglyceridemia, two key components of nephrotic syndrome, have yetto be established. Therapy designed to reduce proteinuria furthercomplicates the study of disease mechanisms. For example,glucocorticoids used to treat proteinuria in MCD independently raiseplasma triglyceride levels (5), and normalization of plasma triglyceridelevels lags behind the response of proteinuria to glucocorticoids incertain forms of nephrotic syndrome, such as MCD (6).

The present disclosure show that increased circulating levels of Angptl4reduce the severity of nephrotic syndrome and conditions associatedtherewith, such as but not limited to, proteinuria. As a result, thepresent disclosure provides method for treating and/or preventingnephrotic syndrome, such as but not limited to, MCD, FSGS, MN/MGN, MPGNand diabetic nephropathy as well as methods of alleviating symptomsassociated with nephrotic syndrome, including, but not limited to,proteinuria and edema. The present disclosure further provides methodsfor reducing proteinuria and edema.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1H shows the development and characterization of aP2-Angptl4 TGrats.

FIG. 1A shows a 2D gel analysis of 200 μg human plasma (n=4patients/group, cropped representative blots shown) and demonstrates thepresence of increased circulating levels of Angptl4 in patients withminimal change disease (MCD) in relapse and in patients with membranousnephropathy (MN) (indicated by arrows), compared to patients with MCD inremission (i. e. non proteinuric patients).

FIG. 1B shows a transgenic (TG) rat model for adipose tissue specificover expression of Angptl4 (aP2-Angplt4 TG).

FIG. 1C shows tissue specific over expression of Angptl4 mRNA (n=3rats/group) in aP2-Angptl4 TG rats. WAT is white adipose tissue, BAT isbrown adipose tissue. ***P<0.001.

FIG. 1D shows 2D gel electrophoresis of 200 μg plasma, followed byWestern blot for Angptl4 and demonstrates that heterozygous aP2-Angptl4TG rats had higher circulating Angptl4 levels than wild type rats (age 3months, n=3 blots/group).

FIG. 1E shows 2D gel electrophoresis of 200 μg plasma, followed byWestern blot with the anti-V5 and anti-Angptl4 antibodies anddemonstrates the presence of adipose tissue secreted V5-tagged Angptl4in the plasma of aP2-Angptl4 TG rats.

FIG. 1F shows 2D gel electrophoresis of anti-N-terminal Angptl4immunoprecipitates from aP2-Angptl4 TG rat plasma followed by Westernblotting using lectin SNA I and anti-Angptl4 antibodies and confirmedthe presence of circulating sialylated Angptl4 in the aP2-Angptl4.

FIG. 1G shows PAS stained sections from 3 month old heterozygousaP2-Angptl4 TG rats (n=3 rats/group) and demonstrates normal glomerularmorphology (magnification 400×).

FIG. 1H shows immunogold EM with anti-V5 antibody to specifically detecttransgenic protein in 3 month heterozygous aP2-Angplt4 TG male rats anddemonstrated gold particles selectively on the endothelial surface inaP2-Angptl4 TG rats (indicated by arrows).

FIGS. 2A-2H shows the relationship of increased circulating levels ofAngptl4 with proteinuria/albuminuria.

FIG. 2A shows assessment of urinary protein excretion (3 μg/lane, exceptMCD remission) in different human and experimental disease conditions byGelCode blue stained SDS PAGE and demonstrated the absence ofsignificant proteinuria in aP2-Angptl4 TG rats (lane marked with *,arrow shows intact albumin at around 70 kDa).

FIG. 2B shows assessment of albuminuria by ELISA and revealed thatheterozygous female aP2-Angptl4 TG rats had lower albuminuria than wildtype littermates (n=6 rats/group).

FIG. 2C shows assessment of albuminuria by ELISA and revealed thatheterozygous male aP2-Angptl4 TG rats had lower albuminuria than wildtype littermates (n=6 rats/group).

FIG. 2D shows induction of puromycin nephrosis (PAN), a model ofnephrotic syndrome, in wild type and aP2-Angptl4 TG rats anddemonstrates less proteinuria in aP2-Angptl4 TG rats compared to wildtype littermates (n=8 rats/group). *P<0.05, **P<0.01 compared tocorresponding controls

FIG. 2E shows recombinant Angptl4 had protective effects on culturedglomerular endothelial cells (GEnCs). **P<0.01, ***P<0.001 compared tocorresponding controls

FIG. 2F shows upregulation of Angptl4 in wild type rats in diseasemodels like PAN on Day 6 was exclusively glomerular, while upregulationof Angptl4 in adipose tissue was noted on Day 10 when proteinuria andglomerular Angptl4 expression are on the decline (n=3 rats/sample).**P<0.01, *** P<0.001 compared to corresponding controls

FIG. 2G shows increased circulating levels of Angptl4 at baseline andafter induction of PAN in aP2-Angptl4 TG rats results in increasedplasma triglyceride levels compared to wild type rats, *P<0.05 comparedto corresponding controls

FIG. 2H shows increased circulating levels of Angptl4 at baseline andafter induction of PAN in aP2-Angptl4 TG rats results in reducedpost-heparin lipoprotein lipase (LPL) activity compared to wild typerats. *P<0.05 compared to corresponding controls

FIG. 3 shows the primers and probes used for Taqman real time PCR (SEQID NOS. 11-22).

FIGS. 4A-4B shows recombinant Angptl4 reduces proteinuria in animalmodels of human glomerular disease.

FIG. 4A shows reduction of proteinuria in Thy1.1 nephritis, a short termmodel of mesangial injury. Thy1.1 nephritis was induced in male Wistarrats (n=4 rats/group). After assessment of baseline proteinuria (Day 1),concentrated supernatant protein from Angptl4 stable or control celllines were injected intra-peritoneally on two consecutive days (Days 1 &2, arrows) into Buffalo Mna rats (n=4 rats/group) followed by assessmentof proteinuria. Proteinuria was lower in Angptl4 treated ratsthroughout, and was statistically significant on Day 5. * P<0.05; **P<0.01. all values are mean±SE.

FIG. 4B shows reduction of proteinuria in Thy1.1 nephritis, a short termmodel of mesangial injury. THY1.1 nephritis was induced in male Wistarrats (n=4 rats/group, injected on Day 0). After confirming the inductionof proteinuria (Day 1), concentrated supernatant protein from Angptl4stable or control cell lines were injected intravenously on twoconsecutive days (Days 1 & 2, arrows)followed by assessment ofproteinuria. Proteinuria was lower in Angptl4 treated rats throughout,and was statistically significant on Day 5. *P<0.05; P<0.01. all valuesare mean±SE

FIGS. 5A-J shows the amino acid and cDNA and peptide sequences ofAngptl4 from various species.

FIG. 5A shows—SEQ ID NOS. 1 and 2: amino acid and cDNA sequence fromhuman (Protein Variant 1 isoform a, long form; underlined amino acidsequences at a position 40 and 161-164).

FIG. 5B shows SEQ ID NOS. 3 and 4: amino acid and cDNA sequence fromhuman (Protein Variant 3 isoform b, short form; underlined amino acidsequences at a position 40 and 161-164).

FIG. 5C shows SEQ ID NOS. 5 and 6: amino acid and cDNA sequence fromrat.

FIG. 5D shows SEQ ID NOS: 7 and 8: amino acid and cDNA from mouse;underlined are forward sequencing primers.

FIG. 5E shows SEQ ID NO. 9: human Protein Variant Derivative with thesequence of interest in generic form. SEQ ID NO.10 shows human ProteinVariant 3 Derivative with the sequence of interest in generic form.

FIGS. 6A-6N shows that elevated circulating Angptl4 levels are requiredfor the development of hypertriglyceridemia in nephrotic syndrome.

FIG. 6A shows ELISA for plasma Angptl4 levels in patients with nephroticsyndrome due to primary glomerular disease. Number of patients analyzedare shown in brackets

FIG. 6B shows ELISA for plasma Angptl4 levels at pre-nephrotic andnephrotic stages in passive Heymann nephritis (PHN, a model ofmembranous nephropathy), Buffalo Mna (B. Mna, spontaneously developfocal and segmental glomerulosclerosis) and single dose intravenouspuromycin aminonucleoside nephrosis (PAN, a model of minimal changedisease), all rat models of nephrotic syndrome.

FIGS. 6C-6E show proteinuria, hypertriglyceridemia and LPL activity inPHN

FIGS. 6F-6H show proteinuria, hypertriglyceridemia and LPL activity inBuffalo Mna rats

FIGS. 6I-6K show proteinuria, hypertriglyceridemia and LPL activity inPAN rats

FIGS. 6L-6M show plasma triglyceride levels and LPL activity in adiposetissue specific Angptl4 overexpressing rats (aP2-Angptl4), that haveelevated circulating Angptl4 levels, and 3 month old podocyte specificAngptl4 overexpressing rats (NPHS2-Angptl4), in which transgeneexpressed Angptl4 does not enter the circulation.

FIG. 6N shows plasma triglyceride levels in Angptl4−/− and +/+ mice 48hours after induction of nephrotic syndrome using γ2-NTS. *P<0.05, **P<0.01, ***P<0.001

FIGS. 7A-7H shows the source of circulating Angptl4 in nephroticsyndrome.

FIG. 7A shows multi-organ Angptl4 mRNA expression relative to control inpassive Heymann nephritis (PHN),

FIG. 7B shows multi-organ Angptl4 mRNA expression relative to control inBuffalo Mna rats and

FIG. 7C shows multi-organ Angptl4 mRNA expression relative to control inpuromycin aminonucleoside nephrosis (PAN).

FIG. 7D shows a representative 2-dimensional gel electrophoresis andWestern blot of plasma showing circulating Angptl4 levels in proteinuricNPHS2-Angptl4 transgenic rats before and after the induction of mild PAN

FIG. 7E shows densitometry analysis of 2-dimensional gels in d

FIG. 7F shows 2-dimensional gel electrophoresis and Western blot ofplasma from NPHS2-Angptl4 transgenic rats with PAN to demonstrate thepresence of V5-tagged transgene expressed Angptl4 in the circulation.

FIGS. 7G AND 7H show plasma triglyceride levels (FIG. 7G) andlipoprotein lipase (LPL) activity (FIG. 7H) six days after induction ofPAN in wild type Sprague Dawley, aP2-Angptl4 and NPHS2-Angptl4transgenic rats. Empty bars correspond to data from FIGS. 6L and 6Mincluded here for comparison. *P<0.05, **P<0.01, ***P<0.001. In panels Gand H, statistical significance is shown for difference betweentransgenic rats and corresponding wild type controls. P<0.001 for eachrat type before and after induction of PAN. In panels A to C, 3-foldchange in expression (horizontal line) was taken as significant.

FIGS. 8A-8E shows urinary loss of Angptl4 and LPL in nephrotic syndrome.

FIG. 8A shows a representative reducing Western blots of urine fromnormal Sprague Dawley (SD), PAN, PHN, and Buffalo Mna rats. Black arrowspoint towards Angptl4 bands. Albumin blush is also noted in PAN, PHN andBuffalo Mna rats between 65 and 70 kDa.

FIG. 8B shows non-reducing Western blot of urine from nephrotic ratsusing goat anti LPL antibody to assess for urinary loss of LPL (arrow).

FIG. 8C shows non-reducing western blot of nephrotic rat urine usinganti-LPL monoclonal antibody 5D2 to identify active LPL (arrow).

FIG. 8D shows a multi-organ mRNA expression profile for LPL in SpragueDawley rats with PAN.

FIG. 8E shows an mRNA expression profile of major organs that expressLPL in aP2-Angptl4 transgenic rats. In panels D and E, 3-fold change inexpression (horizontal line) was taken as significant.

FIGS. 9A-9D shows effect of circulating Angptl4 on proteinuria. Redarrows indicate time points when an antibody or recombinant protein, asappropriate, were injected.

FIG. 9A shows proteinuria after induction of puromycin aminonucleosidenephrosis (PAN) in wild type Sprague Dawley and aP2-Angptl4 transgenicrats

FIG. 9B shows the effect of depleting circulating Angptl4 using ananti-Angptl4 Ab. on proteinuria in Sprague Dawley rats with PAN.

FIG. 9C shows proteinuria in Buffalo Mna rats after injection ofconcentrated supernatant from recombinant rat Angptl4 secreting stablecell lines or control stable cell lines.

FIG. 9D shows the effect of injecting recombinant rat Angptl4 or controlprotein from the above cell lines on proteinuria in severe anti-Thy1.1nephritis, a model of mesangial injury. *P<0.05, **P<0.01

FIGS. 10A-10E shows dissociation of effects of Angptl4 on triglyceridesand proteinuria using mutant recombinant human Angptl4.

FIG. 10A shows a schematic representation of wild type and mutant humanAngptl4 proteins showing mutations in areas important for LPL binding(amino acid 40, and adjacent amino acid 39) and protein cleavage (aminoacids 161 to 164).

FIG. 10B shows a western blot of recombinant tagged proteins using mouseanti V5 antibody and control mouse IgG to demonstrate the expected sizeof the intact protein and reduced cleavage in the mutant proteins(arrows).

FIG. 10C shows plasma levels of wild type or mutant human Angptl4 afterinjecting 55 μg of recombinant human protein in Buffalo Mna rats, amodel of focal and segmental glomerulosclerosis (FSGS) as assessed by OD450 using reagents from the human Angptl4 ELISA kit.

FIG. 10D shows the effect of wild type and mutant Angptl4 on proteinuriain Buffalo Mna rats. *P<0.05, shown where all 3 study groups wereindividually different from control.

FIG. 10E shows the effect of wild type and mutant Angptl4 on plasmatriglyceride levels in Buffalo Mna rats. *P<0.05 for wild type valuescompared to baseline. #P<0.05 for 6 hour mutant values compared to wildtype.

FIGS. 11A-11H shows that circulating Angptl4 reduces proteinuria via itsinteraction with glomerular endothelial αvβ5 integrin. Red arrowsindicate time points when anti β5 integrin antibody or pre-immune serumwere injected.

FIG. 11A shows a confocal image of a glomerulus from an aP2-Angptl4transgenic rat demonstrates co-localization of Angptl4-V5 (anti-V5antibody, red) secreted from adipose tissue with glomerular endothelium(anti Von Willebrand factor antibody, green)

FIG. 11B shows an immunogold electron micrograph of a glomerulus fromaP2-Angptl4 transgenic rats using anti-V5 antibody to show glomerularendothelial cell surface binding of adipose tissue secreted Angptl4-V5.

FIG. 11C shows silaylated Angpt14 protein protected cultured ratglomerular endothelial cells (GEnCs) from oxidative injury, whereashyposialylated Angpt14 increases the effects of oxidative injury.

FIG. 11D shows the interaction of purified αvβ5 integrin with sialylatedAngptl4 or control in vitro. Linear regression slope (black) issuperimposed.

FIG. 11E shows the development of proteinuria after induction ofnephrotic syndrome using γ2-nephrotoxic serum (NTS) in β5 integrin −/−and +/+ mice to demonstrate the protective effects of the endothelial β5integrin—circulating Angptl4 interaction on proteinuria during theperipheral phase of Angptl4 secretion.

FIG. 11F shows the effect of blocking the endothelial β5integrin—Angptl4 interaction using anti β5 integrin antibodies onrecovery from peak proteinuria (corresponds with the peripheral phase ofAngptl4 secretion) in Sprague Dawley rats with PAN.

FIG. 11G shows the effect of blocking the endothelial β5integrin—Angptl4 interaction using anti β5 integrin antibodies onrecovery from peak proteinuria in aP2-Angptl4 transgenic rats with PAN.

FIG. 11H shows the induction of nephrotic syndrome using γ2-NTS inAngptl4−/− and +/+ mice to determine whether the lack of Angptl4 affectsrecovery from peak proteinuria during the peripheral phase of Angptl4expression. The findings of the glomerular phase are consistent with ourpreviously published study (ref. 6 from working example 4). *P<0.05,**P<0.01, ***P<0.001

FIGS. 12A and 12B shows a two-dimensional gel electrophoresis of humanplasma.

FIG. 12A shows a cropped image of representative Western blots withanti-Angptl4 antibody to show elevated circulating Angptl4 levels inmembranous nephropathy (MN), focal and segmental glomerulosclerosis(FSGS) and minimal change disease (MCD). Angptl4 spots are enclosed ingreen circles/ovals.

FIG. 12B shows ponceau red stained images of nitrocellulose membranescorresponding to Western blots.

FIG. 13A shows albuminuria in Angptl4+/+ and Angptl4−/− mice 48 hoursafter injection of γ2-NTS, corresponds to FIG. 6(n). Image is reproducedfrom the on line supplement of Reference 6 from Working Example 4.Similar results seen on Day 2 of study shown in FIG. 6(h).

FIG. 13B shows ponceau red stained images of nitrocellulose membranesused for Western blot in FIG. 2 d.

FIG. 13C shows overexposed Western blot of stripped membrane from FIG. 3b using normal goat serum.

FIG. 13D shows overexposed Western blot of stripped membrane from FIG. 3c using normal mouse IgG.

FIG. 13E shows fasting plasma triglyceride levels in Buffalo Mna ratsfrom 12 hours to 17 days after injection of recombinant human wild typeAngptl4, human mutant Angptl4, and control protein. *P<0.05

FIG. 14A shows elevated plasma Angptl4 levels during the peripheralphase of Angptl4 expression (Days 5 and 7) in γ2-NTS injected ltgb+/+and −/− mice shown in FIG. 11E.

FIG. 14B shows multiorgan mRNA expression profile for Angptl4 in ltgb+/+mice shown in FIG. 11E, 7 days after injection of γ2-NTS.

FIG. 14C shows plasma Angptl4 levels in Sprague Dawley PAN rats shown inFIG. 11F.

FIG. 14D shows plasma triglyceride levels in Sprague Dawley PAN ratsshown in FIG. 11F. All comparisons in panels A, C and D were made withDay 0 values. *P<0.05

FIG. 15 shows a schematic representation of the origin and biologicaleffects of circulating Angptl4 in nephrotic syndrome. Following theestablishment of moderate to severe proteinuria, skeletal muscle,adipose tissue and heart upregulate and secrete Angptl4 into thecirculation. Some of this Angptl4 binds to αvβ5 integrin on theglomerular endothelial surface and reduces proteinuria, while some bindsto, and converts active lipoprotein lipase (LPL) into inactive LPL, thatare lost in urine. Reduced LPL mediated triglyceride uptake results inhypertriglyceridemia. Some circulating Angptl4 is also lost in theurine. In minimal change disease (MCD), podocyte secreted hyposialylatedAngptl4 exerts local pro-proteinuric effects within the glomerulus,whereas podocyte secreted sialylated protein binds the glomerularendothelium and leaks into the circulation to inducehypertriglyceridemia. In membranous nephropathy (MN), focal andsegmental glomerulosclerosis (FSGS) and non-HIV collapsingglomerulopathy (CG), podocytes do not contribute significant amounts ofAngptl4 to the circulation.

FIG. 16A shows a standard curve for human Angptl4 ELISA

FIG. 16B shows a standard curve for rodent Angptl4 ELISA

FIG. 16C shows the characterization of anti-β5 integrin antibody 8472Aby Western blot.

FIG. 16D shows a confocal image of a rat glomerulus that shows bindingof anti-β5 integrin antibody (red, detected using donkey anti rabbitIgG) to glomerular endothelium (blue, labeled with mouse anti-PECAM1antibody) six hours after intravenous injection, resulting in a magentaoverlap pattern.

FIG. 17 shows amino acid and nucleic acid substitutions for four mutantforms of human Angptl4.

FIG. 18 show a Western blot of recombinant proteins shown in FIG. 17tagged using mouse anti-V5 antibody and control mouse IgG to demonstratethe expected size of the intact protein and reduced cleavage in themutant proteins (arrows).

FIG. 19 shows peak levels for the mutant 8511 of change in proteinuriaafter a single intravenous injection into proteinuric Buffalo Mna rats.

FIG. 20 shows decline in proteinuria after injection of 2 mutant humanAngptl4 proteins (15 μg) into Zucker Diabetic Fatty rats, a model ofdiabetic nephropathy and diabetic kidney disease.

FIG. 21 shows levels of proteinuria of two mutant human Angptl4 proteinsafter injection (15 μg) into Zucker Diabetic Fatty rats. All * valuesare relative to baseline Day 0 values.

FIG. 22 shows no increase in plasma triglyceride levels after injectionof 2 mutant forms of human Angptl4 protein (15 μg) in Zucker DiabeticFatty rats. For FIGS. 20-22 : *P<0.05, **P<0.01. #P<0.05.

SUMMARY OF THE DISCLOSURE

In a first aspect, the present disclosure provides methods of treatmentand/or prevention of nephrotic syndrome. In one embodiment, thenephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN ordiabetic nephropathy. In another embodiment, the nephrotic syndrome ischaracterized as MCD. In a further embodiment, the nephrotic syndrome ischaracterized as MSGS. In a further embodiment, the nephrotic syndromeis caused by a diabetic condition. In one embodiment, the diabeticcondition is diabetic nephropathy, diabetes mellitus, lupus nephritis orprimary glomerular disease. The methods comprise the step ofadministering to a subject an Angptl4 polypeptide or an Angptl4polypeptide derivative. In one embodiment, the Angptl4 polypeptidecomprises the sequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 10. In analternate, the amino acid sequence is a fragment of any of the foregoingsequences having an activity comparable to wild type Angptl4 or anAngptl4 polypeptide derivative. In still a further embodiment, theAngptl 4 polypeptide derivative is a derivative described herein and hasbeen modified to have decreased LPL inhibitory activity, to be resistantto cleavage, or a combination of the foregoing. The Angptl4 polypeptideor polypeptide derivative, in one embodiment, is sialylated. Suchderivative may be based on any of the Angplt4 polypeptides describedherein. The Angptl4 polypeptide or polypeptide derivative may beadministered at a therapeutically effective dose, either alone, as apart of a pharmaceutical composition or in combination with a secondaryagent. In one embodiment, such administration treats nephrotic syndromeby providing Angptl4 function. In an alternate embodiment, suchadministration treats nephrotic syndrome by providing a modified Angptl4function, such as, but not limited to, an Angptl4 function that displayreduced LPL inhibition or is resistant to cleavage.

In a second aspect, the present disclosure provides methods of treatmentand/or prevention of MCD. The methods comprise the step of administeringto a subject an Angptl4 polypeptide or an Angptl4 polypeptidederivative. In one embodiment, the Angptl4 polypeptide comprises thesequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 10. In an alternate, the aminoacid sequence is a fragment of any of the foregoing sequences having anactivity comparable to wild type Angptl4. In still a further embodiment,the Angptl 4 polypeptide derivative is a derivative described herein andhas been modified to have decreased LPL inhibitory activity, to beresistant to cleavage, or a combination of the foregoing. The Angptl4polypeptide or polypeptide derivative, in one embodiment, is sialylated.Such derivative may be based on any of the Angplt4 polypeptidesdescribed herein. The Angptl4 polypeptide or polypeptide derivative maybe administered at a therapeutically effective dose, either alone, as apart of a pharmaceutical composition or in combination with a secondaryagent. In one embodiment, such administration treats MCD by providingAngptl4 function. In an alternate embodiment, such administration treatsMCD by providing a modified Angptl4 function, such as, but not limitedto, an Angptl4 function that display reduced LPL inhibition or isresistant to cleavage.

In a third aspect, the present disclosure provides methods ofalleviating one or more symptoms of nephrotic syndrome, such as, but notlimited to, proteinuria, hypercholesterolemia, hypertriglyceridemia andedema. In one embodiment, the nephrotic syndrome is characterized asMCD, FSGS, MN/MGN, MPGN and diabetic nephropathy. In another embodiment,the nephrotic syndrome is characterized as MCD. In a further embodiment,the nephrotic syndrome is caused by FSGS. In a further embodiment, thenephrotic syndrome is caused by a diabetic condition. In one embodiment,the diabetic condition is diabetic nephropathy, diabetes mellitus, lupusnephritis or primary glomerular disease. The methods comprise the stepof administering to a subject an Angptl4 polypeptide or an Angptl4polypeptide derivative. In one embodiment, the Angptl4 polypeptidecomprises the sequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 10. In analternate, the amino acid sequence is a fragment of any of the foregoingsequences having an activity comparable to wild type Angptl4. In still afurther embodiment, the Angptl 4 polypeptide derivative is a derivativedescribed herein and has been modified to have decreased LPL inhibitoryactivity, to be resistant to cleavage, or a combination of theforegoing. The Angptl4 polypeptide or polypeptide derivative, in oneembodiment, is sialylated. Such derivative may be based on any of theAngplt4 polypeptides described herein. The Angptl4 polypeptide orpolypeptide derivative may be administered at a therapeuticallyeffective dose, either alone, as a part of a pharmaceutical compositionor in combination with a secondary agent. In one embodiment, suchadministration alleviates one or more symptoms of nephrotic syndrome byproviding Angptl4 function. In an alternate embodiment, suchadministration alleviates one or more symptoms of nephrotic syndrome byproviding a modified Angptl4 function, such as, but not limited to, anAngptl4 function that display reduced LPL inhibition or is resistant tocleavage.

In a fourth aspect, the present disclosure provides methods for reducingproteinuria in a subject. In one embodiment, the subject is sufferingfrom nephrotic syndrome. In one embodiment, the nephrotic syndrome ischaracterized as MCD, FSGS, MN/MGN, MPGN and diabetic nephropathy. Inanother embodiment, the nephrotic syndrome is characterized as MCD. Inanother embodiment, the subject is suffering from a disordercharacterized by proteinuria. In another embodiment, the subject issuffering from a diabetic condition. In a further embodiment, theproteinuria is caused by FSGS. In one embodiment, the diabetic conditionis diabetic nephropathy, diabetes mellitus, lupus nephritis or primaryglomerular disease. The methods comprise the step of administering to asubject an Angptl4 polypeptide or an Angptl4 polypeptide derivative. Inone embodiment, the Angptl4 polypeptide comprises the sequence of SEQ IDNOS: 1, 3, 5, 7, 9 or 10. In an alternate, the amino acid sequence is afragment of any of the foregoing sequences having an activity comparableto wild type Angptl4. In still a further embodiment, the Angptl 4polypeptide derivative is a derivative described herein and has beenmodified to have decreased LPL inhibitory activity, to be resistant tocleavage, or a combination of the foregoing. The Angptl4 polypeptide orpolypeptide derivative, in one embodiment, is sialylated. Suchderivative may be based on any of the Angplt4 polypeptides describedherein. The Angptl4 polypeptide or polypeptide derivative may beadministered at a therapeutically effective dose, either alone, as apart of a pharmaceutical composition or in combination with a secondaryagent. In one embodiment, such administration reduces proteinuria byproviding Angptl4 function. In an alternate embodiment, suchadministration reduces proteinuria by providing a modified Angptl4function, such as, but not limited to, an Angptl4 function that displayreduced LPL inhibition or is resistant to cleavage.

In a fifth aspect, the present disclosure provides methods of reducingedema in a subject. In one embodiment, the subject is suffering fromnephrotic syndrome. In one embodiment, the nephrotic syndrome ischaracterized as MCD, FSGS, MN/MGN, MPGN, and diabetic nephropathy. Inanother embodiment, the nephrotic syndrome is characterized as MCD. In afurther embodiment, the nephrotic syndrome is caused by FSGS. In aspecific embodiment, the edema is caused by decreased circulating levelsof plasma proteins such as albumin. In a further embodiment, thenephrotic syndrome is caused by a diabetic condition In one embodiment,the diabetic condition is diabetic nephropathy, diabetes mellitus, lupusnephritis or primary glomerular disease. Reduction of proteinuriathrough the administration of an Angptl4 polypeptide of Angptl4polypeptide derivative will reduce proteinuria, raise plasma proteinlevels and thereby reduce edema. The methods comprise the step ofadministering to a subject an Angptl4 polypeptide or an Angptl4polypeptide derivative. In one embodiment, the Angptl4 polypeptidecomprises the sequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 10. In analternate, the amino acid sequence is a fragment of any of the foregoingsequences having an activity comparable to wild type Angptl4. In still afurther embodiment, the Angptl4 polypeptide derivative is a derivativedescribed herein and has been modified to have decreased LPL inhibitoryactivity, to be resistant to cleavage, or a combination of theforegoing. The Angptl4 polypeptide or polypeptide derivative, in oneembodiment, is sialylated. Such derivative may be based on any of theAngplt4 polypeptides described herein. The Angptl4 polypeptide orpolypeptide derivative may be administered at a therapeuticallyeffective dose, either alone, as a part of a pharmaceutical compositionor in combination with a secondary agent. In one embodiment, suchadministration reduces edema by providing Angptl4 function. In analternate embodiment, such administration reduces edema by providing amodified Angptl4 function, such as, but not limited to, an Angptl4function that display reduced LPL inhibition or is resistant tocleavage.

In a sixth aspect, the present disclosure provides methods of reducinghypercholesterolemia and/or hypertriglyceridemia in a subject. In oneembodiment, the subject is suffering from nephrotic syndrome. In oneembodiment, the nephrotic syndrome is characterized as MCD, FSGS,MN/MGN, MPGN and diabetic nephropathy. In another embodiment, thenephrotic syndrome is characterized as MCD. In a further embodiment, thenephrotic syndrome is caused by a diabetic condition In one embodiment,the diabetic condition is diabetic nephropathy, diabetes mellitus, lupusnephritis or primary glomerular disease. The methods comprise the stepof administering to a subject an Angptl4 polypeptide or an Angptl4polypeptide derivative. In one embodiment, the Angptl4 polypeptidecomprises the sequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 10. In analternate, the amino acid sequence is a fragment of any of the foregoingsequences having an activity comparable to wild type Angptl4. In still afurther embodiment, the Angptl 4 polypeptide derivative is a derivativedescribed herein and has been modified to have decreased LPL inhibitoryactivity, to be resistant to cleavage, or a combination of theforegoing. The Angptl4 polypeptide or polypeptide derivative, in oneembodiment, is sialylated. Such derivative may be based on any of theAngplt4 polypeptides described herein, The Angptl4 polypeptide orpolypeptide derivative may be administered at a therapeuticallyeffective dose, either alone, as a part of a pharmaceutical compositionor in combination with a secondary agent. In one embodiment, suchadministration reduces hypercholesterolemia and/or hypertriglyceridemiaby providing Angptl4 function. In an alternate embodiment, suchadministration reduces hypercholesterolemia and/or hypertriglyceridemiaby providing a modified Angptl4 function, such as, but not limited to,an Angptl4 function that display reduced LPL inhibition or is resistantto cleavage.

In a seventh aspect, the present disclosure provides methods oftreatment and/or prevention of a diabetic condition. In one embodiment,the diabetic condition is diabetic nephropathy, diabetes mellitus, lupusnephritis or primary glomerular disease. The methods comprise the stepof administering to a subject an Angptl4 polypeptide or an Angptl4polypeptide derivative. In one embodiment, the Angptl4 polypeptidecomprises the sequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 10. In analternate, the amino acid sequence is a fragment of any of the foregoingsequences having an activity comparable to wild type Angptl4. In still afurther embodiment, the Angptl 4 polypeptide derivative is a derivativedescribed herein and has been modified to have decreased LPL inhibitoryactivity, to be resistant to cleavage, or a combination of theforegoing. The Angptl4 polypeptide or polypeptide derivative, in oneembodiment, is sialylated. Such derivative may be based on any of theAngplt4 polypeptides described herein, The Angptl4 polypeptide orpolypeptide derivative may be administered at a therapeuticallyeffective dose, either alone, as a part of a pharmaceutical compositionor in combination with a secondary agent. In one embodiment, suchadministration treats the foregoing conditions by providing Angptl4function. In an alternate embodiment, such administration treats theforegoing conditions by providing a modified Angptl4 function, such as,but not limited to, an Angptl4 function that display reduced LPLinhibition or is resistant to cleavage.

In an eighth aspect, the present disclosure provides a pharmaceuticalcomposition for use in the methods of the first through sixth aspects.The composition comprises one or more Anptl4 polypeptides or polypeptidederivatives. In one embodiment, the Angptl4 polypeptide comprises thesequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 10. In an alternate, the aminoacid sequence is a fragment of any of the foregoing sequences having anactivity comparable to wild type Angptl4. In still a further embodiment,the Angptl 4 polypeptide derivative is a derivative described herein andhas been modified to have decreased LPL inhibitory activity, to beresistant to cleavage, or a combination of the foregoing. The Angptl4polypeptide or polypeptide derivative, in one embodiment, is sialylated.Such derivative may be based on any of the Angplt4 polypeptidesdescribed herein.

DETAILED DESCRIPTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

While investigating nephrotic syndrome, it was noted that Angptl4secreted from podocytes induced proteinuria. More importantly, asdescribed herein, circulating Angptl4 reduced the proteinuria in atransgenic animal model. Increased levels of Angptl 4 have been noted innephrotic syndrome, such as MCD and MN, but increased circulating levelsof Angptl4 have not been associated with causation of nephroticsyndrome.

While increased Angptl4 levels are shown to treat nephrotic syndrome andreduce associated proteinuria, increased Angptl4 in the circulation hasbeen observed to induce hyperlipidemia (hypertriglyceridemia), such as,but not limited to, through inhibition of LPL. It would be advantageousto provide the benefits of increased circulating Angptl4 levels withoutthe negative consequences of hyperlipidemia. Such an approach ispossible using the Angptl4 polypeptide derivatives as disclosed herein.

Angiopoietin-like proteins have been implicated in the development ofhypertriglyceridemia and tumor metastasis, and are functionally distinctfrom the angiopoietins. Angptl4 is a PPAR (8) and PPAR (9) target genehighly expressed in the liver and adipose tissue, strongly induced byfasting in white adipose tissue and liver, and is an apoptosis survivalfactor for vascular endothelial cells under normoxic conditions (10).Angptl4 is a potent inhibitor of LPL (11), inducing significanthypertriglyceridemia following intravenous injection oradenovirus-mediated expression (12, 13). Other studies showed lesserexpression of Angptl4 in cardiomyocytes and skeletal muscle, and lowlevel expression in whole kidney on Northern blot analysis (8). Recentpopulation based studies of the ANGPTL4 gene reveals variants thataffect triglyceride levels in humans (14, 15).

The present disclosure shows a conclusive role for circulating Angptl4in the reduction of proteinuria observed in nephrotic syndrome, such as,but not limited to, MCD, FSGS, MN, MPGN and diabetic nephropathy.

Definitions

The terms “prevention”, “prevent”, “preventing”, “suppression”,“suppress” and “suppressing” as used herein refer to a course of action(such as administering a compound or pharmaceutical composition)initiated prior to the onset of a symptom, aspect, or characteristics ofa disease or condition so as to prevent or reduce such symptom, aspect,or characteristics. Such preventing and suppressing need not be absoluteto be useful.

The terms “treatment”, “treat” and “treating” as used herein refers acourse of action (such as administering a compound or pharmaceuticalcomposition) initiated after the onset of a symptom, aspect, orcharacteristics of a disease or condition so as to eliminate or reducesuch symptom, aspect, or characteristics. Such treating need not beabsolute to be useful.

The term “in need of treatment” as used herein refers to a judgment madeby a caregiver that a patient requires or will benefit from treatment.This judgment is made based on a variety of factors that are in therealm of a caregiver's expertise, but that includes the knowledge thatthe patient is ill, or will be ill, as the result of a disease orcondition that is treatable by a method or compound of the disclosure.

The term “in need of prevention” as used herein refers to a judgmentmade by a caregiver that a patient requires or will benefit fromprevention. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that includes the knowledgethat the patient will be ill or may become ill, as the result of adisease or condition that is preventable by a method or compound of thedisclosure.

The term “individual”, “subject” or “patient” as used herein refers toany animal, including mammals, such as mice, rats, other rodents,rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, andhumans. The term may specify male or female or both, or exclude male orfemale.

The term “therapeutically effective amount” as used herein refers to anamount of a compound, either alone or as a part of a pharmaceuticalcomposition, that is capable of having any detectable, positive effecton any symptom, aspect, or characteristics of a disease or condition.Such effect need not be absolute to be beneficial. When referring to anAngptl4 polypeptide or Angptl4 polypeptide derivative, the term“therapeutically effective amount” refers to an amount of suchpolypeptide sufficient to reduce proteinuria in a subject.

The term “pharmaceutically acceptable derivative” means anypharmaceutically acceptable salt, ester, salt of an ester, solvate orother derivative of an Angptl4 polypeptide or polypeptide derivative ofthe present disclosure that, upon administration to a subject, iscapable of providing (directly or indirectly) the function of wild typeAngptl4; in certain embodiment, the Angptl4 polypeptide or polypeptidederivative shows decreased LPL inhibitory activity of a resistance tocleavage. Particularly favored derivatives are those that increase thebioavailability of an Angptl4 polypeptide or polypeptide derivative ofthe disclosure when such polypeptides are administered to a subject(e.g., by allowing an orally administered compound to be more readilyabsorbed into the blood), enhance delivery of such polypeptides to agiven biological compartment, increase solubility to allowadministration by injection, alter metabolism or alter rate ofexcretion. In one embodiment, the derivative is a prodrug.

The term “pharmaceutically acceptable salt(s)”, unless otherwiseindicated, includes salts of acidic or basic groups that may be presentin the Angptl4 polypeptide or polypeptide derivative of the presentdisclosure.

The terms “about” and “approximately” shall generally mean an acceptabledegree of error or variation for the quantity measured given the natureor precision of the measurements. Typical, exemplary degrees of error orvariation are within 20 percent (%), preferably within 10%, and morepreferably within 5% of a given value or range of values. For biologicalsystems, the term “about” refers to an acceptable standard deviation oferror, preferably not more than 2-fold of a give value. Numericalquantities given herein are approximate unless stated otherwise, meaningthat the term “about” or “approximately” can be inferred when notexpressly stated.

Methods of Treatment and Prevention

The present disclosure provides methods of treatment and/or preventionof nephrotic syndrome. The present disclosure further provides methodsof treatment and/or prevention of MCD, FSGS, and/or conditions withmesangial injury (such as diabetes mellitus). The present disclosurefurther provides methods of treatment and/or prevention of a diabeticcondition. In one embodiment, the diabetic condition is diabeticnephropathy, diabetes mellitus, lupus nephritis or primary glomerulardisease. The present disclosure additionally provides methods ofalleviating one or more symptoms of nephritic syndrome, such as, but notlimited to, proteinuria, hypercholesterolemia, hypertriglyceridemia andedema. Still further, the present disclosure provides for methods ofreducing proteinuria. Further still, the present disclosure providesmethods of reducing edema. The present disclosure additionally providesfor pharmaceutical compositions comprising one or more Angptl4polypeptides of Angptl4 polypeptide derivatives. The nature of theAngptl4 polypeptide derivatives is described in further detail below.

In one embodiment, the teachings of the present disclosure provide forthe treatment and/or prevention of nephrotic syndrome in a subject inneed of such treatment or prevention. In one embodiment, the nephroticsyndrome is characterized as MCD, FSGS, MN/MGN, and MPGN. In anotherembodiment, the nephrotic syndrome is characterized as MCD. In a furtherembodiment, the nephrotic syndrome is caused by FSGS. In a furtherembodiment, the nephrotic syndrome is caused by a diabetic condition. Inone embodiment, the diabetic condition is diabetic nephropathy, diabetesmellitus, lupus nephritis or primary glomerular disease. The methodscomprise the step of administering to a subject an Angptl4 polypeptideor an Angptl4 polypeptide derivative. In one embodiment, the Angptl4polypeptide comprises the sequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 10.In an alternate, the amino acid sequence is a fragment of any of theforegoing sequences having an activity comparable to wild type Angptl4.In still a further embodiment, the Angptl 4 polypeptide derivative is aderivative described herein and has been modified to have decreased LPLinhibitory activity, to be resistant to cleavage, or a combination ofthe foregoing. The Angptl4 polypeptide or polypeptide derivative, in oneembodiment, is sialylated. Such derivative may be based on any of theAngplt4 polypeptides described herein. The Angptl4 polypeptide orpolypeptide derivative may be administered at a therapeuticallyeffective dose, either alone, as a part of a pharmaceutical compositionor in combination with a secondary agent. In one embodiment, suchadministration treats nephrotic syndrome by providing Angptl4 function.In an alternate embodiment, such administration treats nephroticsyndrome by providing a modified Angptl4 function, such as, but notlimited to, an Angptl4 function that display reduced LPL inhibition oris resistant to cleavage. Such method may further comprise identifying asubject in need of such treatment and/or prevention.

In an alternate embodiment, the teachings of the present disclosureprovide for the treatment and/or prevention of MCD in a subject in needof such treatment or prevention. The methods comprise the step ofadministering to a subject an Angptl4 polypeptide or an Angptl4polypeptide derivative. In one embodiment, the Angptl4 polypeptidecomprises the sequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 10. In analternate, the amino acid sequence is a fragment of any of the foregoingsequences having an activity comparable to wild type Angptl4. In still afurther embodiment, the Angptl4 polypeptide derivative is a derivativedescribed herein and has been modified to have decreased LPL inhibitoryactivity, to be resistant to cleavage, or a combination of theforegoing. The Angptl4 polypeptide or polypeptide derivative, in oneembodiment, is sialylated. Such derivative may be based on any of theAngplt4 polypeptides described herein. The Angptl4 polypeptide orpolypeptide derivative may be administered at a therapeuticallyeffective dose, either alone, as a part of a pharmaceutical compositionor in combination with a secondary agent. In one embodiment, suchadministration treats MCD by providing Angptl4 function. In an alternateembodiment, such administration treats MCD by providing a modifiedAngptl4 function, such as, but not limited to, an Angptl4 function thatdisplay reduced LPL inhibition or is resistant to cleavage. Such methodmay further comprise identifying a subject in need of such treatmentand/or prevention.

In further embodiment, the teachings of the present disclosure providefor methods of alleviating one or more symptoms of nephrotic syndrome,such as, but not limited to, proteinuria, hypercholesterolemia,hypertriglyceridemia and edema. In one embodiment, the nephroticsyndrome is characterized as MCD, FSGS, MN/MGN, MPGN, and diabeticnephropathy. In another embodiment, the nephrotic syndrome ischaracterized as MCD. In a further embodiment, the nephrotic syndrome iscaused by FSGS. In a further embodiment, the nephrotic syndrome iscaused by a diabetic condition. In one embodiment, the diabeticcondition is diabetic nephropathy, diabetes mellitus, lupus nephritis orprimary glomerular disease. The methods comprise the step ofadministering to a subject an Angptl4 polypeptide or an Angptl4polypeptide derivative. In one embodiment, the Angptl4 polypeptidecomprises the sequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 10. In analternate, the amino acid sequence is a fragment of any of the foregoingsequences having an activity comparable to wild type Angptl4. In still afurther embodiment, the Angptl 4 polypeptide derivative is a derivativedescribed herein and has been modified to have decreased LPL inhibitoryactivity, to be resistant to cleavage, or a combination of theforegoing. The Angptl4 polypeptide or polypeptide derivative, in oneembodiment, is sialylated. Such derivative may be based on any of theAngplt4 polypeptides described herein. The Angptl4 polypeptide orpolypeptide derivative may be administered at a therapeuticallyeffective dose, either alone, as a part of a pharmaceutical compositionor in combination with a secondary agent. In one embodiment, suchadministration alleviates one or more symptoms of nephrotic syndrome byproviding Angptl4 function. In an alternate embodiment, suchadministration alleviates one or more symptoms of nephrotic syndrome byproviding a modified Angptl4 function, such as, but not limited to, anAngptl4 function that display reduced LPL inhibition or is resistant tocleavage. Such method may further comprise identifying a subject in needof such treatment and/or prevention.

In still a further embodiment, the teachings of the present disclosureprovide methods for reducing proteinuria in a subject. In oneembodiment, the subject is suffering from nephrotic syndrome. In oneembodiment, the nephrotic syndrome is characterized as MCD, FSGS,MN/MGN, MPGN and diabetic nephropathy. In another embodiment, thenephrotic syndrome is characterized as MCD, In a further embodiment, thenephrotic syndrome is caused by FSGS. In a further embodiment, thenephrotic syndrome is caused by a diabetic condition. In one embodiment,the diabetic condition is diabetic nephropathy, diabetes mellitus, lupusnephritis or primary glomerular disease. The methods comprise the stepof administering to a subject an Angptl4 polypeptide or an Angptl4polypeptide derivative. In one embodiment, the Angptl4 polypeptidecomprises the sequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 10. In analternate, the amino acid sequence is a fragment of any of the foregoingsequences having an activity comparable to wild type Angptl4. In still afurther embodiment, the Angptl 4 polypeptide derivative is a derivativedescribed herein and has been modified to have decreased LPL inhibitoryactivity, to be resistant to cleavage, or a combination of theforegoing. The Angptl4 polypeptide or polypeptide derivative, in oneembodiment, is sialylated. Such derivative may be based on any of theAngplt4 polypeptides described herein. The Angptl4 polypeptide orpolypeptide derivative may be administered at a therapeuticallyeffective dose, either alone, as a part of a pharmaceutical compositionor in combination with a secondary agent. In one embodiment, suchadministration reduces proteinuria by providing Angptl4 function. In analternate embodiment, such administration reduces proteinuria byproviding a modified Angptl4 function, such as, but not limited to, anAngptl4 function that display reduced LPL inhibition or is resistant tocleavage. Such method may further comprise identifying a subject in needof such treatment and/or prevention.

In yet a further embodiment, the teachings of the present disclosureprovide methods for reducing edema in a subject. In one embodiment, thesubject is suffering from nephrotic syndrome. In one embodiment, thenephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN anddiabetic nephropathy. In another embodiment, the nephrotic syndrome ischaracterized as MCD. In a further embodiment, the nephrotic syndrome iscaused by FSGS. In a further embodiment, the nephrotic syndrome iscaused by a diabetic condition. In one embodiment, the diabeticcondition is diabetic nephropathy, diabetes mellitus, lupus nephritis orprimary glomerular disease. In a specific embodiment, the edema iscaused by decreased circulating levels of plasma proteins such asalbumin. Reduction of proteinuria through the administration of anAngptl4 polypeptide or a Angptl4 polypeptide derivative will raisereduce proteinuria, raise plasma protein levels and thereby reduceedema. The methods comprise the step of administering to a subject anAngptl4 polypeptide or an Angptl4 polypeptide derivative. In oneembodiment, the Angptl4 polypeptide comprises the sequence of SEQ IDNOS: 1, 3, 5, 7, 9 or 10. In an alternate, the amino acid sequence is afragment of any of the foregoing sequences having an activity comparableto wild type Angptl4. In still a further embodiment, the Angptl 4polypeptide derivative is a derivative described herein and has beenmodified to have decreased LPL inhibitory activity, to be resistant tocleavage, or a combination of the foregoing. The Angptl4 polypeptide orpolypeptide derivative, in one embodiment, is sialylated. Suchderivative may be based on any of the Angplt4 polypeptides describedherein. The Angptl4 polypeptide or polypeptide derivative may beadministered at a therapeutically effective dose, either alone, as apart of a pharmaceutical composition or in combination with a secondaryagent. In one embodiment, such administration reduces edema by providingAngptl4 function. In an alternate embodiment, such administrationreduces edema by providing a modified Angptl4 function, such as, but notlimited to, an Angptl4 function that display reduced LPL inhibition oris resistant to cleavage. Such method may further comprise identifying asubject in need of such treatment and/or prevention.

In still a further embodiment, the teachings of the present disclosureprovide methods for reducing hypercholesterolemia and/orhypertriglyceridemia in a subject. In one embodiment, the subject issuffering from nephrotic syndrome. In one embodiment, the nephroticsyndrome is characterized as MCD, FSGS, MN/MGN, and MPGN. In anotherembodiment, the nephrotic syndrome is characterized as MCD. In a furtherembodiment, the nephrotic syndrome is caused by FSGS. In a furtherembodiment, the nephrotic syndrome is caused by a diabetic condition. Inone embodiment, the diabetic condition is diabetic nephropathy, diabetesmellitus, lupus nephritis or primary glomerular disease. The methodscomprise the step of administering to a subject an Angptl4 polypeptideor an Angptl4 polypeptide derivative. In one embodiment, the Angptl4polypeptide comprises the sequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 10.In an alternate, the amino acid sequence is a fragment of any of theforegoing sequences having an activity comparable to wild type Angptl4.In still a further embodiment, the Angptl 4 polypeptide derivative is aderivative described herein and has been modified to have decreased LPLinhibitory activity, to be resistant to cleavage, or a combination ofthe foregoing. The Angptl4 polypeptide or polypeptide derivative, in oneembodiment, is sialylated. Such derivative may be based on any of theAngplt4 polypeptides described herein. The Angptl4 polypeptide orpolypeptide derivative may be administered at a therapeuticallyeffective dose, either alone, as a part of a pharmaceutical compositionor in combination with a secondary agent. In one embodiment, suchadministration reduces proteinuria by providing Angptl4 function. In analternate embodiment, such administration reduces proteinuria byproviding a modified Angptl4 function, such as, but not limited to, anAngptl4 function that display reduced LPL inhibition or is resistant tocleavage. Such method may further comprise identifying a subject in needof such treatment and/or prevention.

In still a further embodiment, the teachings of the present disclosureprovide methods for treatment and/or prevention of a nephrotic syndromethat is caused by a diabetic condition. In one embodiment, the diabeticcondition is diabetic nephropathy, diabetes mellitus, lupus nephritis orprimary glomerular disease. The methods comprise the step ofadministering to a subject an Angptl4 polypeptide or an Angptl4polypeptide derivative. In one embodiment, the Angptl4 polypeptidecomprises the sequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 10. In analternate, the amino acid sequence is a fragment of any of the foregoingsequences having an activity comparable to wild type Angptl4. In still afurther embodiment, the Angptl 4 polypeptide derivative is a derivativedescribed herein and has been modified to have decreased LPL inhibitoryactivity, to be resistant to cleavage, or a combination of theforegoing. The Angptl4 polypeptide or polypeptide derivative, in oneembodiment, is sialylated. Such derivative may be based on any of theAngplt4 polypeptides described herein, The Angptl4 polypeptide orpolypeptide derivative may be administered at a therapeuticallyeffective dose, either alone, as a part of a pharmaceutical compositionor in combination with a secondary agent. In one embodiment, suchadministration treats the foregoing conditions by providing Angptl4function. In an alternate embodiment, such administration treats theforegoing conditions by providing a modified Angptl4 function, such as,but not limited to, an Angptl4 function that display reduced LPLinhibition or is resistant to cleavage.

Some embodiments of administering the Angptl4 polypeptide or derivativeinvolve a form of administration that delivers the polypeptide to theblood. In one example the polypeptide is administered intravenously.Given the appropriate dosage form, such administration may be performedorally, subcutaneously, or by other means as is known in the art. TheAngptl4 polypeptide or derivative may be administered in atherapeutically effective amount; this amount will generally be within acertain range of ratios of mass of compound to mass of subject. In someembodiments of the method the polypeptide is administered at a dosage ofabout 0.005-150,000 μg/kg, 0.5-15,000 μg/kg, 5-1500 μg/kg, or 50-150μg/kg. Thus, for a typical 70 kg human adult, the dosage may be0.0035-11,000 mg, 0.035-1100 mg, 0.35-110 mg, or 3.5-11 mg.Administration may occur on a regular schedule. In some embodiments ofthe method the polypeptide is administered about once per 14 days. Inother embodiments the polypeptide is administered about twice per month.In still other embodiments the polypeptide is administered from aboutonce per month to about twice per month. In further embodiments, thepolypeptide is administered once per a given time period selected fromthe group consisting of: a day, two days, three days, a week, ten days,two weeks, three weeks, four weeks, and a month.

Methods of Screening

The present disclosure also relates to a method for identifying acompound effective for treating or preventing nephrotic syndrome or acondition associated therewith, such as, but not limited to,proteinuria, hypercholesterolemia, hypertriglyceridemia or edema. In oneembodiment, the nephrotic syndrome is characterized as MCD or MN. Inanother embodiment, the nephrotic syndrome is characterized as MCD. Inanother embodiment, the nephrotic syndrome is characterized by FSGS. Ina further embodiment, the nephrotic syndrome is caused by a diabeticcondition. In one embodiment, the diabetic condition is diabeticnephropathy, diabetes mellitus, lupus nephritis or primary glomerulardisease. Such compounds may be useful as active ingredients included inpharmaceutical compositions or for administration alone. In oneembodiment, the methods include determining the level a polypeptideinvolved in the etiology of nephrotic syndrome, such as, but not limitedto, Angptl4.

In general, such screening methods comprises the steps of providing anassay system (as described in more detail below) that expresses apolypeptide involved in the etiology of nephrotic syndrome, such as, butnot limited to, Angptl4, introducing into the assay system a testcompound to be tested and determining whether the effect of the testcompound on the level the polypeptide. The methods involve theidentification of candidate or test compounds or agents (polypeptides,functional nucleic acids, carbohydrates, antibodies, small molecules orother molecules) which effect the level of sialylation of thepolypeptide. Such compounds may then be further tested in appropriatesystems (such as, but not limited to, the animal models systemsdescribed herein) to determine the activity of the identified compounds.

Candidate compounds are identified using a variety of assays, such as,but not limited to, assays that employ cells which express a polypeptideinvolved in the etiology of nephrotic syndrome, such as, but not limitedto, Angptl4 or in assays with isolated polypeptides. The various assayscan employ a variety of variants of such polypeptides (e. g.,full-length, a biologically active fragment, or a fusion protein whichincludes all or a portion of the desired polypeptide). Moreover, suchpolypeptides can be derived from any suitable mammalian species (e. g.,human, rat or murine); in a specific embodiment, the polypeptide isderived from a human.

Where the assay involves the use of a whole cell, the cell may eithernaturally express a polypeptide involved in the etiology of nephroticsyndrome, such as, but not limited to, Angptl4, or may be modified toexpress the same. In the latter case, cells can be modified to express adesired polypeptide through conventional molecular biology techniques,such as by infecting the cell with a virus comprising such polypeptide.The cell can also be a prokaryotic or an eukaryotic cell that has beentransfected with a nucleotide sequence encoding such polypeptide. In theforegoing, full length polypeptides, fragments or fusion proteinscontaining at least a part of such polypeptide may be used. Exemplaryassay systems are described in the current specification.

The various screening assays may be combined with an in vivo assayentailing measuring the effect of the test compound on the symptoms thedisease states and conditions discussed herein. In such an embodiment,the compounds may be evaluated to determine if they impact a parameterassociated with nephrotic syndrome or a condition related thereto, suchas, but not limited to, proteinuria or edema. Such parameters include,but are not limited to, determining 1) the level of a polypeptideinvolved in the etiology of nephrotic syndrome and related conditions,such as, but not limited to Angptl4 and 2) determining the level ofprotein excretion, either total or with regard to specific components.

In one embodiment, such a screening assay can be performed, for example,by determining the level of a polypeptide, such as, but not limited to,Angptl4 and detecting a difference in the level of such polypeptide inthe presence of as compared to the absence of a test compound. Suchscreening assay may be in vitro, in vivo or ex vivo and may be cellculture based (either with whole cells or lysates) or may be based on ananimal model. Any assay of the present disclosure may be used in theforegoing method.

Suitable test compounds for use in the screening methods can be obtainedfrom any suitable source, such as conventional compound libraries. Thetest compounds can also be obtained using any of the numerous approachesin combinatorial library methods known in the art, including: biologicallibraries, spatially addressable parallel solid phase or solution phaselibraries, synthetic library methods requiring deconvolution, the“one-bead one-compound” library method and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds. Examples of methods for the synthesis of molecularlibraries can be found in the art. Libraries of compounds may bepresented in solution or on beads, bacteria, spores, plasmids or phage.

The present disclosure also provides kits for carrying out any method ofthe present disclosure, which can contain any of the compounds and/orcompositions disclosed herein or otherwise useful for practicing amethod of the disclosure.

Creation and Selection of Angptl4 Polypeptide Derivatives

Angiopoietin-related protein 4 is a polypeptide that in humans isencoded by the ANGPTL4 gene. This gene is a member of theangiopoietin/angiopoietin-like gene family and encodes a glycosylated,secreted protein with a N-terminal signal sequence (amino acid residues1-22 of SEQ ID NO:1), a coiled-coil domain (amino acid residues 23-170of SEQ ID NO:1), a linker region (amino acid residues 171-185 of SEQ IDNO:1) and a fibrinogen C-terminal domain (amino acid residues 186-406 ofSEQ ID NO:1). This gene is induced under hypoxic conditions inendothelial cells and is the target of peroxisome proliferationactivators. The encoded protein is a serum hormone directly involved inregulating glucose homeostasis, lipid metabolism, and insulinsensitivity and also acts as an apoptosis survival factor for vascularendothelial cells. Alternatively spliced transcript variants encodingdifferent isoforms have been described. This gene was previouslyreferred to as ANGPTL2 but has been renamed ANGPTL4

Angptl4 inhibits LPL by breaking the LPL dimer molecule. Angptl4 hasbeen unambiguously established as potent inhibitors of blood plasmatriglyceride (TG) clearance, causing elevation of plasma TG levels.Recent evidence indicates that variations in the sequence of the Angptl4polypeptide impact the effect on triglycerides, with certain mutationsconferring reduced triglyceride levels implying a decreased inhibitionof LPL (33 and 34, each of which are incorporated by reference for theteaching of Angptl4 variants). Furthermore, it has been reported thatAngptl4 polypeptides exist in oligomeric forms and that oligomerizationis required for inhibition of LPL activity. Once secreted from the cell,the oligomeric form is cleaved at a cleavage site (R₁₆₁RKR₁₆₄ of SEQ IDNOS: 1 and 3) to provide monomeric C-terminal forms and oligomericN-terminal forms (34). The N-terminal residues 1-187 of the Angptl4peptide were found to be sufficient to inhibit LPL (33).

The amino acid and cDNA sequences of the human, rat and mouse areprovided in FIGS. 5A-F and designated SEQ ID NOS: 1-8. The presentdisclosure contemplates the use of Angptl4 polypeptides and polypeptidederivatives in the methods disclosed herein, such as but not limited to,methods of treatment and prevention. As defined herein an Angptl4polypeptide derivative refers to an Angptl4 polypeptide that includesone or more insertions, deletions and/or substitutions as determinedfrom the amino acid sequence of the human polypeptides shown in SEQ IDNOS: 1 or 3 or the polypeptides shown in SEQ ID NOS: 5 or 7.

Some embodiments of the Angptl14 derivative comprise a core structurethat is a consensus sequence between any two or more of SEQ ID NOS: 1,3, 5, or 7, with one or more substitutions as described herein. Oneembodiment of the Angptl14 derivative comprises the consensus sequencebetween SEQ ID NOS: 1 and 3 (both version of human Angptl14); theconsensus sequence comprising:A-B-Cin which A is at least 80% homologous to SEQ ID NO: 26, B is anoligopeptide of 0-38 residues (an optional linking region), and C is atleast 80% homologous to SEQ ID NO: 27. The level of homology of A to SEQID NO: 26 and of C to SEQ ID NO: 27 may of course be higher than 80%.These levels of homology may be independently selected from 80-100%, forexample 85%, 90%, 95%, 99%, 99.5%, and 100%. The sequence ofoligopeptide B may be any sequence. Some embodiments of oligopeptide Bare at least 50% homologous to positions 184-222 of SEQ ID NO: 1. Insuch embodiments the level of homology may be selected from any point inthe range of 50-100%, including for exemplary purposes 60%, 70%, 80%,85%, 90%, 95%, 99%, 99.5%, and 100%.

Another embodiment of the Angptl14 derivative comprises a consensussequence between all of SEQ ID NOS: 1, 3, 5, AND 7 (human variant, ratand mouse); the consensus sequence comprising:V-W-X-Y-Zin which V has at least 80% homology to SEQ ID NO: 23, W is anoligopeptide of 0-5 residues, X has at least 80% homology to SEQ ID NO:24, Y is an oligopeptide of 0-38 residues (an optional linking region),and Z has at least 80% homology to SEQ ID NO: 25. The levels of homologyof V to SEQ ID NO: 23, X to SEQ ID NO 24, and Z to SEQ ID NO: 25 may behigher than 80%. These levels of homology may be independently selectedfrom 80-100%, for example 85%, 90%, 95%, 99%, 99.5%, and 100%. Thesequence of oligopeptide Y may be any sequence. Some embodiments ofoligopeptide B are at least 50% homologous to positions 184-222 of SEQID NO: 1. In such embodiments the level of homology may be selected fromany point in the range of 50-100%, including for exemplary purposes 60%,70%, 80%, 85%, 90%, 95%, 99%, 99.5%, and 100%.

These consensus sequences allow for substitutions at positionscorresponding to positions 39, 40, 46, 50, and 53 of SEQ ID NO: 1, whichas taught in this disclosure may serve to reduce LPL inhibitoryactivity. They also allow for substitutions at positions correspondingto positions 63-66 of SEQ ID NO: 1, which as taught in this disclosuremay serve to increase the protein's resistance to cleavage. They alsoallow for substitutions at positions corresponding to SEQ ID NO: 1positions 5, 67, 72, 77, 167, 174, 190, 230, 233, 237, 251, 266, 278,291, 293, 296, 307, 308, 336, 338, 349, 361, 371, and 384, as these wererevealed to be sites of known natural human variants by a search onUniProt (www.uniprot.orq). Specific embodiments of the Angptl14derivative comprise one or more of the following substitutions at thesepositions: P5L, S67R, R72L, G77R, E167K, P174S, E190Q, E196K, R230C,G233R, F237V, P251T, T266M, R278Q, V291M, L293M, E296V, P307S, V308M,R336C, D338E, W349C, G361R, G361S, R371Q, and R384W. Such naturallyoccurring substitutions would be expected to preserve the function ofthe protein.

In one embodiment, amino acid residues of the Angptl4 polypeptide areremoved and replaced with different amino acid residues. The variantsmay be constructed as described herein or as known in the art. Thevariants so constructed may be evaluated using the methods and assaysdescribed herein to screen for activity.

When used herein, single letters when used to refer to amino acids havethe following meanings:

G Glycine P Proline W Tryptophan H Histidine A Alanine V Valine K LysineR Arginine L Leucine I Isoleucine Q Glutamine N Asparagine M MethionineC Cysteine E Glutamic Acid D Aspartic Acid F Phenylalanine Y Tyrosine SSerine T Threonine

In one embodiment, the variant comprises a change in the amino acidsequence of an Angptl4 polypeptide that decreases the ability of Angptl4to inhibit LPL or to or to be resistant to cleavage. The change may be areplacement, deletion and/or substitution of one or more residues inthis region. Such changes have been described in the art (see references33 and 34 which are herein incorporated by reference for such teaching).In one embodiment, such change occurs in residues 1-187 with respect toSEQ ID NO: 1, residues 1-182 of SEQ ID NO: 3, residues 1-182 of SEQ IDNO: 26, any residues in SEQ ID NO: 23, and residues 1-79 in SEQ ID NO:24.

Some embodiments of the derivative of the Angptl4 polypeptide derivativediffer from the human wild-type sequence at positions 39-55 of SEQ IDNO: 1 (DEMNVLAHGLLQLGQGL); this region corresponds to positions 39-55 ofSEQ ID NOS: 1, 3, 5, 7, 23, and 26. Additional embodiments of thederivative comprise a sequence at positions 39-55 that is neitherDEMNVLAHGLLQLGQGL (positions 39-55 of SEQ ID NO: 1) norDKMNVLAHGLLQLGQGL (SEQ ID NO: 28). Further embodiments of the Angptl4polypeptide derivative have at least one substitution at positions 39,40, 46, 50, and 53, such that positions 39-40 of V is not DE, position46 of V is not H, position 50 of V is not Q, and position 53 of V is notQ.

In some embodiments, such change occurs at position 40 with respect toSEQ ID NOS: 1, 3, 5, 7, 23, or 26. In one embodiment, the amino acid atposition 40 (a negatively charged glutamic acid residue in wild-typeAngptl4) is replaced with a neutral amino acid or a positively chargedamino acid. In a particular embodiment, the change is an E40Ksubstitution. In another particular embodiment, the change is an E40Asubstitution. The E40K and E40A substitutions have been shown to reduceLPL inhibition by Angptl4, but not interfere with expression, secretion,processing and other functions of the polypeptide. In a furtherparticular embodiment, the change at position 40 is selected from thoseshown in Table 1 below. In yet a further embodiment, the amino acid atposition 39 of SEQ ID NOS: 1, 3, 5, 7, 23, or 26 (a negatively chargedaspartic acid residue in wild-type Angptl4) is replaced with a neutralor positively charged amino acid. In one embodiment, the substitution isa D39K substitution of a D39A substitution. In a further particularembodiment, the change at position 39 of SEQ ID NOS: 1, 3, 5, 7, 23, or26 is selected from those shown in Table 1 below. In certainembodiments, a polypeptide variant may contain one of the aforementionedchanges at position 40, one of the aforementioned changes at position 39or a combination of the foregoing. In a particular embodiment, thepolypeptide contains a D39K substitution and a E40K substitution, a D39Asubstitution and a E40K substitution or a D39K substitution and an E40Asubstitution. In a further specific embodiment the polypeptidederivative the sequence at positions 39-40 is selected from the groupconsisting of: DK, KE, DA, and AE. In yet another embodiment thepolypeptide derivative the sequence at positions 39-40 is not DE.

TABLE 1 Modifications of D₃₉ and E₄₀ in the human Angptl4 protein G P VL I M C F Y W H R Q N S T

In another embodiment, the derivative comprises one or more changes in aregion of the Angptl4 polypeptide responsible for cleavage of thepolypeptide. In one embodiment, this region is the R₁₆₁RKR₁₆₄ region ofAngptl4 (corresponding to positions 161-164 of SEQ ID NOS: 1, 3, 5, 7,and 26; and positions 63-66 of SEQ ID NO: 24). The change may be areplacement, deletion and/or substitution of one or more residues inthis region. The R₁₆₁RKR₁₆₄ region has been shown to be responsible forcleavage of the oligomeric forms of Angptl4, releasing oligomers of theN-terminal sequences and monomers of the C-terminal sequence. Forms ofAngptl4 with a mutated cleavage site were shown to accumulate at higherlevels in the circulation than wild-type polypeptide. Furthermore,preventing cleavage of the Angptl4 polypeptide stabilizes the oligomericforms of Angptl4 observed to be efficacious in the present disclosure.In one embodiment, all 4 amino acid residues of the R₁₆₁RKR₁₆₄ regionare changed, such that the sequence is not RRKR; in an alternateembodiment, any 1, 2 or 3 amino acid residues of the R₁₆₁RKR₁₆₄ regionare changed. In a further embodiment, the arginine residues at positions161, 162 or 164 are independently substituted with glycine, alanine,valine or serine and the lysine residue at position 163 is substitutedwith glycine, alanine, valine or serine. In a specific embodiment theR₁₆₁RKR₁₆₄ sequence is replaced with a sequence selected from the groupconsisting of: GAAG (SEQ ID NO: 29), GSGS (SEQ ID NO: 80), GVVA (SEQ IDNO: 49), SGGG (SEQ ID NO: 87), and VAVA (SEQ ID NO: 90). In a furtherspecific embodiment the R₁₆₁RKR₁₆₄ sequence is replaced with AAVV.Exemplary amino acid sequences for replacement of the entire R₁₆₁RKR₁₆₄region of SEQ ID NOS: 1 or 3 is provided in Table 2 below.

TABLE 2Modifications of ₁₆₁RRKR₁₆₄ in the Angptl4 protein or derivatives SEQSEQ SEQ SEQ SEQ SEQ SEQ ID ID ID ID ID ID ID 29 GAAG 38 GAAV 47 GAVV 56GVVV 65 GAAA 74 AVVV 83 SGSG 30 GAGA 39 GAVA 48 GVAV 57 VGVV 66 AGAA 75VAVV 84 SGGS 31 GGAA 40 GVAA 49 GVVA 58 VVVG 67 AAAG 76 VVVV 85 SSGG 32AGGA 41 AGVA 50 AGVV 59 VVGV 68 AAGA 77 SSSS 86 GSGG 33 AGAG 42 AGAV 51AVVG 60 VAVG 69 AAVV 78 GGGG 87 SGGG 34 AAGG 43 AAVG 52 AVGV 61 VVGA 70AAVA 79 AAAA 88 GGSG 35 VGAA 44 AAGV 53 VGAV 62 VVAG 71 AAAV 80 GSGS 89GGGS 36 VAAG 45 AVAG 54 VGVA 63 VVVA 72 AVAA 81 GSSG 90 VAVA 37 VAGA 46AVGA 55 VAGV 64 VVAV 73 VAAA 82 GGSS

In a further embodiment, one or more of the amino acids in theR₁₆₁RKR₁₆₄ sequence is altered to remove a consensus binding site of anenzyme capable of cleaving Angplt4, such that Angptl4 is resistant tocleavage. In one embodiment, the enzyme is a proprotein convertase andthe consensus binding site is RXKR, RXRR, RR or KR, where X is any aminoacid. In making such alternations, one or more amino acids may bedeleted or substituted with glycine, alanine, valine or serine or withany of the other substitutions discussed herein.

In still a further embodiment, the variant comprises one or more changesin a region of the Angptl4 polypeptide responsible for oligomerizationof the polypeptide. In one embodiment, this region is the C₇₆ and/or C₈₀region of Angptl4. The C₇₆ and/or C₈₀ region has been shown to beinvolved in oligomerization of the Angptl4 polypeptide (34, whichreference is incorporated herein for such teaching). The change may be areplacement, deletion and/or substitution of one or more residues inthis region. In a particular embodiment, only one of the cysteineresidues at positions 76 and 80 is substituted; in an alternateembodiment, both cysteine residues at positions 76 and 80 are bothsubstituted. In one embodiment, at least one of the cysteine residues atposition 76 and 80 are substituted independently with alanine or serine;in another embodiment, both cysteine residues are substituted withalanine or serine.

In a further embodiment, the variant comprises one or more changes inthe R₁₆₁RKR₁₆₄ region of Angplt4 that inhibits the cleavage of theAngptl4 polypeptide oligomer and a change at position 40 that reducesinhibition of LPL activity by Angptl4. Any of the changes discussedherein are included.

In one embodiment, the present disclosure provides for Angptl4polypeptide variants having the amino acid sequence of SEQ ID NOS: 9 or10. SEQ ID NO: 9 is shown in FIG. 4 and includes the wild type sequenceof Angptl4 from SEQ ID NO: 1, with the exception of substitutions atpositions 39, 40, 76, 80 and 161-164 indicated by X₃₉, X₄₀, X₇₆, X₈₀,X₁₆₁, X₁₆₂, X₁₆₃ and X₁₆₄, respectively. SEQ ID NO: 10 is shown in FIG.4 and includes the wild type sequence of Angptl4 from SEQ ID NO: 3, withthe exception of substitutions at positions 39, 40, 76, 80 and 161-164indicated by X₃₉, X₄₀, X₇₆, X₈₀, X₁₆₁, X₁₆₂, X₁₆₃ and X₁₆₄,respectively.

In SEQ ID NOS: 9 and 10, X₃₉ may be A, G, P, V, L, I, M, C, F, Y, W, H,R, Q, N, S, T or K. In one embodiment, X₃₉ is a neutral or positivelycharged amino acid. In a further embodiment, X₃₉ may be A or K. In stilla further embodiment, X₃₉ may be D.

In SEQ ID NOS: 9 and 10, X₄₀ may be A, G, P, V, L, I, M, C, F, Y, W, H,R, Q, N, S, T or K. In one embodiment, X₄₀ is a neutral or positivelycharged amino acid. In a further embodiment, X₄₀ may be A or K. In stilla further embodiment, X₄₀ may be E. In yet a further embodiment, X₄₀ maybe E when X₃₉ is not D and X₃₉ may be D when X₄₀ is not E.

In SEQ ID NOS: 9 and 10, at least one of X₇₆ and X₈₀ may be substituted.In one embodiment, X₇₆ and X₈₀ are independently A or S or C. In oneembodiment, one of X₇₆ and X₈₀ may be A or S and the other of X₇₆ andX₈₀ is C. In a further embodiment, both of X₇₆ and X₈₀ may beindependently A or S. In still a further embodiment, both of X₇₆ and X₈₉may C.

In SEQ ID NOS: 9 and 10, at least one of X₁₆₁, X₁₆₂, X₁₆₃ and X₁₆₄ maybe substituted. In one embodiment, all 4 of X₁₆₁, X₁₆₂, X₁₆₃ and X₁₆₄are substituted; in an alternate embodiment, 1, 2 or 3 of X₁₆₁, X₁₆₂,X₁₆₃ and X₁₆₄ are substituted. In a further embodiment, X₁₆₁, X₁₆₂, X₁₆₃and X₁₆₄ are independently D, R, K, G, A, V or S. In still a furtherembodiment, all 4 of are substituted with the combinations recited inTable 2.

The present disclosure contemplates combinations of the foregoing in anyform. Furthermore, the designated residues in SEQ ID NOS: 9 and 10 maybe substituted with conservative amino acid substitutions as designatedin Table 3, or with residues having a difference in hydropathic index of+/−1 or less or with residues having a difference in hydrophilicityvalues of +/−1 or less.

In a one embodiment, X₃₉ is D, X₄₀ is A or K, X₇₆ and X₈₀ are C andX₁₆₁, X₁₆₂, X₁₆₃ and X₁₆₄ are independently substituted with D, R, K, G,A, V or S, optionally provided that at least one of X₁₆₁, X₁₆₂, X₁₆₃ andX₁₆₄ is an amino acid not found in SEQ ID NOS: 1 or 3. In anotherembodiment, X₃₉ is D, X₄₀ is A or K, X₇₆ and X₈₀ are C and X₁₆₁, X₁₆₂,X₁₆₃ and X₁₆₄ are selected from the combinations shown in Table 2. Instill another embodiment, X₃₉ is D, X₄₀ is A or K, X₇₆ and X₈₀ are C andX₁₆₁, X₁₆₂, X₁₆₃ and X₁₆₄ are GSGS or GAAG.

In an additional embodiment, X₉ is D, X₄₀ is A or K, one of X₇₆ and X₈₀is A or S and the other of X₇₆ and X₈₀ is C and X₁₆₁, X₁₆₂, X₁₆₃ andX₁₆₄ are independently substituted with D, R, K, G, A, V or S,optionally provided that at least one of X₁₆₁, X₁₆₂, X₁₆₃ and X₁₆₄ is anamino acid not found in SEQ ID NOS: 1 or 3. In a further embodiment, X₃₉is D, X₄₀ is A or K, one of X₇₆ and X₈₀ is A or S and the other of X₇₆and X₈₀ is C and X₁₆₁, X₁₁₆₂, X₁₆₃ and X₁₆₄ are selected from thecombinations shown in Table 2. In still a further embodiment, X₃₉ is D,X₄₀ is A or K, one of X₇₆ and X₈₀ is A or S and the other of X₇₆ and X₈₀is C and X₁₆₁, X₁₆₂, X₁₆₃ and X₁₆₄ are GSGS or GAAG.

In one embodiment, X₃₉ is A or K, X₄₀ is E, X₇₆ and X₈₀ are C and X₁₆₁,X₁₆₂, X₁₆₃ and X₁₆₄ are independently substituted with D, R, K, G, A, Vor S, optionally provided that at least one of X₁₆₁, X₁₆₂, X₁₆₃ and X₁₆₄is an amino acid not found in SEQ ID NOS: 1 or 3. In another embodiment,X₃₉ is A or K, X₄₀ is E, X₇₆ and X₈₀ are C and X₁₆₁, X₁₆₂, X₁₆₃ and X₁₆₄are selected from the combinations shown in Table 2. In still anotherembodiment, X₃₉ is A or K, X₄₀ is E, X₇₆ and X₈₀ are C and X₁₆₁, X₁₆₂,X₁₆₃ and X₁₆₄ are GSGS or GAAG.

In one embodiment, X₃₉ is D, X₄₀ is K, X₇₆ and X₈₀ are C and X₁₆₁, X₁₆₂,X₁₆₃ and X₁₆₄ are independently substituted with D, R, K, G, A, V or S,optionally provided that at least one of X₁₆₁, X₁₆₂, X₁₆₃ and X₁₆₄ is anamino acid not found in SEQ ID NOS: 1 or 3. In another embodiment, X₃₉is D, X₄₀ is K, X₇₆ and X₈₀ are C and X₁₆₁, X₁₆₂, X₁₆₃ and X₁₆₄ areselected from the combinations shown in Table 2, In still anotherembodiment, X₃₉ is D, X₄₀ is K, X₇₆ and X₈₀ are C and X₁₆₁, X₁₆₂, X₁₆₃and X₁₆₄ are GSGS or GAAG.

In one embodiment, X₃₉ is 0, X₄₀ is K, one of X₇₆ and X₈₀ is A or S andthe other of X₇₆ and X₈₀ is C and X₁₆₁, X₁₆₂, X₁₆₃ and X₁₆₄ areindependently substituted with D, R, K, G, A, V or S, optionallyprovided that at least one of X₁₆₁, X₁₆₂, X₁₆₃ and X₁₆₄ is an amino acidnot found in SEQ ID NOS: 1 or 3. In another embodiment, X₃₉ is D, X₄₀ isK, one of X₇₆ and X₈₀ is A or S and the other of X₇₆ and X₈₀ is C andX₁₆₁, X₁₆₂, X₁₆₃ and X₁₆₄ are selected from the combinations shown inTable 2. In still another embodiment, X₃₉ is D, X₄₀ is K, one of X₇₆ andX₈₀ is A or S and the other of X₇₆ and X₈₀ is C and X₁₆₁, X₁₆₂, X₁₆₃ andX₁₆₄ are GSGS or GAAG.

In one embodiment, the Angptl4 derivative is based on a fragment ofAngplt4. Suitable fragments include any fragment that retains theactivity of wild type Angplt4 or any fragment of 100 or more consecutiveamino acids. In one embodiment, such fragment is based on amino acids1-187 SEQ ID NO: 1 or amino acids 1-182 of SEQ ID NO: 3. Such fragmentsmay have the amino acid substitutions described in the precedingparagraphs.

The Angptl4 polypeptide derivative may have an activity that iscomparable to or increased (in one embodiment, 50% or more) as comparedto the wild-type Angptl4 polypeptide activity; alternatively, theAngptl4 polypeptide derivative may have an activity that is decreased(in one embodiment, less than 50%) as compared to the wild-type Angptl4polypeptide activity. In a specific embodiment, the Angptl4 polypeptidederivative has a decreased ability to inhibit LPL and shows an increasedresistance to cleavage.

The deletions, additions and substitutions can be selected, as would beknown to one of ordinary skill in the art, to generate a desired Angptl4polypeptide derivative. For example, conservative substitutions orsubstitutions of amino acids with similar properties are expected to betolerated. In addition, specific deletions, insertions and substitutionsmay impact, positively or negatively, a certain Angptl4 polypeptideactivity but not impact a different Angptl4 polypeptide activity.

Conservative modifications to the amino acid sequence of any of SEQ IDNOS: 1 or 3 or 5 or 7, including combinations thereof (and thecorresponding modifications to the encoding nucleotides) will produceAngptl4 polypeptide derivatives having functional and chemicalcharacteristics similar to those of naturally occurring Angptl4polypeptides while minimizing undesirable properties such as LPLinhibitory activity. In contrast, substantial modifications in thefunctional and/or chemical characteristics of Angptl4 polypeptides maybe accomplished by selecting substitutions in the amino acid sequence ofany of SEQ ID NOS: 1 or 3 or 5 or 7, including combinations thereof,that differ significantly in their effect on maintaining (a) thestructure of the molecular backbone in the area of the substitution.

For example, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a nonnative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine.

Conservative amino acid substitutions also encompass non-naturallyoccurring amino acid residues which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics, and other reversed or invertedforms of amino acid moieties. It will be appreciated by those of skillin the art that nucleic acid and polypeptide molecules described hereinmay be chemically synthesized as well as produced by recombinant means.

Naturally occurring residues may be divided into classes based on commonside chain properties: 1) hydrophobic: norleucine, Met, Ala, Val, Leu,Ile; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 3) acidic: Asp,Glu; 4) basic: His, Lys, Arg; 5) residues that influence chainorientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange ofa member of one of these classes for a member from another class. Suchsubstituted residues may be introduced into regions of the Angptl4polypeptide derivatives that are homologous with non-human Angptl4polypeptide orthologs, or into the non-homologous regions of themolecule.

In making such changes, the hydropathic index of amino acids may beconsidered, Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics, these are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1,8); glycine(−0.4); threonine (−0,7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art(Kyte et al., J. Mol. Biol., 157: 105-131, 1982). It is known thatcertain amino acids may be substituted for other amino acids having asimilar hydropathic index or score and still retain a similar biologicalactivity.

In making changes based upon the hydropathic index, the substitution ofamino acids whose hydropathic indices are within +/−2 may be used; in analternate embodiment, the hydropathic indices are with +/−1; in yetanother alternate embodiment, the hydropathic indices are within +/−0.5.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. Thegreatest local average hydrophilicity of a polypeptide as governed bythe hydrophilicity of its adjacent amino acids, correlates with abiological property of the protein.

The following hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+−0.1);glutamate (+3.0.+−0.1); serine (+0.3); asparagine (+0.2); glutamine(+0.2); glycine (0); threonine (−0.4); proline (−0.5.+−0.1); alanine(−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine(−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5); tryptophan (−3.4).

In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within +/−2may be used; in an alternate embodiment, the hydrophilicity values arewith +/−1; in yet another alternate embodiment, the hydrophilicityvalues are within +/−0.5.

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the Angptl4polypeptide, or to increase or decrease the affinity of the Angptl4polypeptide with a particular binding target in order to increase ordecrease an Angptl4 polypeptide activity.

Exemplary amino acid substitutions are set forth in Table 3.

TABLE 3 Amino Acid Substitutions Original Preferred Amino Acid Exemplarysubstitution substitution Ala Val, Leu, Ile Val Arg Lys, Gln, Asn LysAsn Glu Glu Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp GlyPro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe,Norleucine Leu Leu Ile, Val, Met, Ala, Phe, Norleucine Ile Lys Arg,1,4-diaminobutyric acid, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu,Val, Ile, Ala, Tyr Leu Pro Ala, Gly Gly Ser Thr, Ala, Cys Thr Thr SerSer Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe,Ala, Norleucine Leu

A skilled artisan will be able to determine suitable variants of thepolypeptide as set forth in any of SEQ ID NOS: 1, 3, 5, 7, 9, 10, and23-27, including combinations thereof, using well known techniques. Foridentifying suitable areas of the molecule that may be changed withoutdestroying activity, one skilled in the art may target areas notbelieved to be important for activity. For example, when similarpolypeptides with similar activities from the same species or from otherspecies are known, one skilled in the art may compare the amino acidsequence of an Angptl4 polypeptide to such similar polypeptides. Withsuch a comparison, one can identify residues and portions of themolecules that are conserved among similar polypeptides. It will beappreciated that changes in areas of an Angptl4 polypeptide that are notconserved relative to such similar polypeptides would be less likely toadversely affect the biological activity and/or structure of the Angptl4polypeptide. One skilled in the art would also know that, even inrelatively conserved regions, one may substitute chemically similaramino acids for the naturally occurring residues while retainingactivity (conservative amino acid residue substitutions). Therefore,even areas that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in an Angptl4 polypeptide thatcorrespond to amino acid residues that are important for activity orstructure in similar polypeptides. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues of an Angptl4 polypeptide.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of that information, one skilled in the art maypredict the alignment of amino acid residues of an Angptl4 polypeptidewith respect to its three dimensional structure. One skilled in the artmay choose not to make radical changes to amino acid residues predictedto be on the surface of the protein, since such residues may be involvedin important interactions with other molecules. Moreover, one skilled inthe art may generate test Angptl4 polypeptide derivatives containing asingle amino acid substitution at each desired amino acid residue. Thederivatives can then be screened using activity assays know to thoseskilled in the art and as disclosed herein. Such derivatives could beused to gather information about suitable substitution. For example, ifone discovered that a change to a particular amino acid residue resultedin destroyed, undesirably reduced, or unsuitable activity, derivativeswith such a change would be avoided. In other words, based oninformation gathered from such routine experiments, one skilled in theart can readily determine the amino acids where further substitutionsshould be avoided either alone or in combination with other mutations.

Numerous scientific publications have been devoted to the prediction ofsecondary structure from analyses of amino acid sequences (see Chou etal., Biochemistry, 13(2): 222-245, 1974; Chou et al., Biochemistry,113(2): 211-222, 1974; Chou et al., Adv. Enzymol. Relat. Areas Mol.Biol., 47: 45-148, 1978; Chou et al., Ann. Rev. Biochem., 47: 251-276,1979; and Chou et al., Biophys. J., 26: 367-384, 1979). Moreover,computer programs are currently available to assist with predictingsecondary structure of polypeptides. Examples include those programsbased upon the Jameson-Wolf analysis (Jameson et al., Comput. Appl.Biosci., 4(1): 181-186, 1998; and Wolf et al., Comput. Appl. Biosci.,4(1): 187-191; 1988), the program PepPlot® (Brutlag et al., CABS, 6:237-245, 1990; and Weinberger et al., Science, 228: 740-742, 1985), andother new programs for protein tertiary structure prediction (Fetrow. etal., Biotechnology, 11: 479-483, 1993).

Moreover, computer programs are currently available to assist withpredicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural data base (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure (see Holmet al., Nucl. Acid, Res., 27(1): 244-247, 1999).

Additional methods of predicting secondary structure include “threading”(Jones, D., Curr Opin. Struct. Biol., 7(3): 377-87, 1997; Suppl et al.,Structure, 4(1): 15-9, 1996), “profile analysis” (Bowie et al., Science,253: 164-170, 1991; Gribskov et al., Meth. Enzym., 183: 146-159, 1990;and Gribskov et al., Proc. Nat. Acad. Sci., 84(13): 4355-4358, 1987),and. “evolutionary linkage” (See Home, supra, and Brenner, supra).

Any of the polypeptide forms discussed herein may also contain asequence useful in the identification or purification of thepolypeptide; an example of such a sequence is the C-terminal V5 tag. Theforegoing also includes nucleic acid sequences (such as, but not limitedto cDNA sequences) coding for such polypeptides, including polypeptidederivatives as described herein.

Compositions

Useful compositions of the present disclosure may comprise one or morepolypeptides of the present disclosure useful in the treatment andprevention methods of the present disclosure; useful compositions alsoinclude one or more nucleic acids coding for one or more polypeptides ofthe present disclosure useful in the treatment and prevention methods ofthe present disclosure. The compositions disclosed may comprise one ormore of such compounds, in combination with a pharmaceuticallyacceptable carrier. Examples of such carriers and methods of formulationmay be found in Remington: The Science and Practice of Pharmacy (20^(th)Ed., Lippincott, Williams & Wilkins, Daniel Limmer, editor). To form apharmaceutically acceptable composition suitable for administration,such compositions will contain an therapeutically effective amount ofcompound.

The pharmaceutical compositions of the disclosure may be used in thetreatment and prevention methods of the present disclosure. Suchcompositions are administered to a subject in amounts sufficient todeliver a therapeutically effective amount of the compound(s) so as tobe effective in the treatment and prevention methods disclosed herein.The therapeutically effective amount may vary according to a variety offactors such as, but not limited to, the subject's condition, weight,sex and age. Other factors include the mode and site of administration.The pharmaceutical compositions may be provided to the subject in anymethod known in the art. Exemplary routes of administration include, butare not limited to, subcutaneous, intravenous, topical, epicutaneous,oral, intraosseous, and intramuscular. The compositions of the presentdisclosure may be administered only one time to the subject or more thanone time to the subject. Furthermore, when the compositions areadministered to the subject more than once, a variety of regimens may beused, such as, but not limited to, one per day, once per week or onceper month. The compositions may also be administered to the subject morethan one time per day. The therapeutically effective amount andappropriate dosing regimens may be identified by routine testing inorder to obtain optimal activity, while minimizing any potential sideeffects. In addition, co-administration or sequential administration ofother agents may be desirable.

The therapeutically effective amount may be a range of ratios betweenthe mass of the compound and the mass of the subject. In someembodiments of the compositions the therapeutically effective amount isabout 0.005-150,000 μg/kg, 0.5-15,000 μg/kg, 5-1500 μg/kg, or 50-150μg/kg. Thus, for a typical 70 kg human adult, the therapeuticallyeffective amount may be 0.0035-11,000 mg, 0.035-1100 mg, 0.35-110 mg, or3.5-11 mg. Administration may occur on a regular schedule.

The compositions of the present disclosure may be administeredsystemically, such as by intravenous administration, or locally such asby subcutaneous injection or by application of a paste or cream. In someembodiments of the composition containing an Angptl4 polypeptide orderivative, the pharmaceutical will be suitable for delivery of thepolypeptide to the blood. Such suitable types of pharmaceuticals includeintravenous formulations, intramuscular formulations, transdermal pastesor creams, transdermal patches, suppositories, and oral dosages formsthat protect the polypeptide from digestion.

In one embodiment, a nucleic acid, which may be in the form of asuitable plasmid or vector, is provided that codes for an Angptl4polypeptide or Angptl4 polypeptide variant of the present disclosure.Such nucleic acid is introduced into a cell, which may be obtained fromthe subject, by suitable methods known in the art (for example,electroporation). In one embodiment, the cell is an adipose cell. Thecells may be assayed for expression of the Angptl4 polypeptide orpolypeptide derivative (in one embodiment, expression of the polypeptidecan be determined by the presence of a tag on the polypeptide asdiscussed herein). The cells expressing an Angptl4 polypeptide ofpolypeptide derivative may then be introduced into the subject. In oneembodiment, the cells are administered to the subject by subcutaneousinjection; other methods of administration may also be used, includingthose discussed herein. The cells then express Angptl4 polypeptide or anAngptl4 polypeptide derivative, which is taken up into the circulation.

The compositions of the present disclosure may further comprise agentswhich improve the solubility, half-life, absorption, etc. of thecompound(s). Furthermore, the compositions of the present disclosure mayfurther comprise agents that attenuate undesirable side effects and/oror decrease the toxicity of the compounds(s). Examples of such agentsare described in a variety of texts, such a, but not limited to,Remington: The Science and Practice of Pharmacy (20^(th) Ed.,Lippincott, Williams & Wilkins, Daniel Limmer, editor).

The compositions of the present disclosure can be administered in a widevariety of dosage forms for administration. For example, thecompositions can be administered in forms, such as, but not limited to,tablets, capsules, sachets, lozenges, troches, pills, powders, granules,elixirs, tinctures, solutions, suspensions, elixirs, syrups, ointments,creams, pastes, emulsions, or solutions for intravenous administrationor injection. Other dosage forms include administration transdermally,via patch mechanism or ointment. Any of the foregoing may be modified toprovide for timed release and/or sustained release formulations.

In the present disclosure, the pharmaceutical compositions may furthercomprise a pharmaceutically acceptable carriers include, but are notlimited to, vehicles, adjuvants, surfactants, suspending agents,emulsifying agents, inert fillers, diluents, excipients, wetting agents,binders, lubricants, buffering agents, disintegrating agents andcarriers, as well as accessory agents, such as, but not limited to,coloring agents and flavoring agents (collectively referred to herein asa carrier), Typically, the pharmaceutically acceptable carrier ischemically inert to the active compounds and has no detrimental sideeffects or toxicity under the conditions of use. The pharmaceuticallyacceptable carriers can include polymers and polymer matrices. Thenature of the pharmaceutically acceptable carrier may differ dependingon the particular dosage form employed and other characteristics of thecomposition.

For instance, for oral administration in solid form, such as but notlimited to, tablets, capsules, sachets, lozenges, troches, pills,powders, or granules, the compound(s) may be combined with an oral,non-toxic pharmaceutically acceptable inert carrier, such as, but notlimited to, inert fillers, suitable binders, lubricants, disintegratingagents and accessory agents. Suitable binders include. withoutlimitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include, without limitation, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, and the like.Disintegrators include, without limitation, starch, methyl cellulose,agar, bentonite, xanthum gum and the like. Tablet forms can include oneor more of the following: lactose, sucrose, mannitol, corn starch,potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acidas well as the other carriers described herein. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin, or sucrose and acadia,emulsions, and gels containing, in addition to the active ingredient,such carriers as are known in the art.

For oral liquid forms, such as but not limited to, tinctures, solutions,suspensions, elixirs, syrups, the nucleic acid molecules of the presentdisclosure can be dissolved in diluents, such as water, saline, oralcohols. Furthermore, the oral liquid forms may comprise suitablyflavored suspending or dispersing agents such as the synthetic andnatural gums, for example, tragacanth, acacia, methylcellulose and thelike. Moreover, when desired or necessary, suitable and coloring agentsor other accessory agents can also be incorporated into the mixture.Other dispersing agents that may be employed include glycerin and thelike.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the patient, and aqueous andnon-aqueous sterile suspensions that can include suspending agents,solubilizers, thickening agents, stabilizers, and preservatives. Thecompound(s) may be administered in a physiologically acceptable diluent,such as a sterile liquid or mixture of liquids, including water, saline,aqueous dextrose and related sugar solutions, an alcohol, such asethanol, isopropanol, or hexadecyl alcohol, glycols, such as propyleneglycol or polyethylene glycol such as poly(ethyleneglycol) 400, glycerolketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, an oil, afatty acid, a fatty acid ester or glyceride, or an acetylated fatty acidglyceride with or without the addition of a pharmaceutically acceptablesurfactant, such as, but not limited to, a soap, an oil or a detergent,suspending agent, such as, but not limited to, pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations, include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includepolyethylene sorbitan fatty acid esters, such as sorbitan monooleate andthe high molecular weight adducts of ethylene oxide with a hydrophobicbase, formed by the condensation of propylene oxide with propyleneglycol, oleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters. Suitablesoaps for use in parenteral formulations include fatty alkali metal,ammonium, and triethanolamine salts, and suitable detergents include (a)cationic detergents such as, for example, dimethyldialkylammoniumhalides, and alkylpyridinium halides, (b) anionic detergents such as,for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether,and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergentssuch as, for example, fatty amine oxides, fatty acid alkanolamides, andpolyoxyethylene polypropylene copolymers, (d) amphoteric detergents suchas, for example, alkylbeta-aminopropionates, and 2-alkylimidazolinequaternary ammonium salts, and (e) mixtures thereof.

Suitable preservatives and buffers can be used in such formulations. Inorder to minimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5% toabout 15% by weight.

Topical dosage forms, such as, but not limited to, ointments, creams,pastes, emulsions, containing the nucleic acid molecule of the presentdisclosure, can be admixed with a variety of carrier materials wellknown in the art, such as, e.g., alcohols, aloe vera gel, allantoin,glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate,and the like, to form alcoholic solutions, topical cleansers, cleansingcreams, skin gels, skin lotions, and shampoos in cream or gelformulations. Inclusion of a skin exfoliant or dermal abrasivepreparation may also be used. Such topical preparations may be appliedto a patch, bandage or dressing for transdermal delivery or may beapplied to a bandage or dressing for delivery directly to the site of awound or cutaneous injury.

The compound(s) of the present disclosure can also be administered inthe form of liposome delivery systems, such as small unilamellarvesicles, large unilamellar vesicles and multilamellar vesicles.Liposomes can be formed from a variety of phospholipids, such ascholesterol, stearylamine or phosphatidylcholines. Such liposomes mayalso contain monoclonal antibodies to direct delivery of the liposome toa particular cell type or group of cell types.

The compound(s) of the present disclosure may also be coupled withsoluble polymers as targetable drug carriers. Such polymers can include,but are not limited to, polyvinyl-pyrrolidone, pyran copolymer,polyhydroxypropylmethacryl-amidephenol,polyhydroxyethylaspartamidephenol, or polyethyl-eneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates andcross-linked or amphipathic block copolymers of hydrogels.

WORKING EXAMPLES

In the following results, the methods used were those methods specifiedin the Methods section of the present disclosure and the referencescited therein. Some of the following results are described in Clement LC et. al., “Podocyte secreted Angiopoietin-like 4 mediates proteinuriain glucocorticoid-sensitive nephrotic syndrome,” Nature Medicine,January 2011 (this reference is hereby incorporated by reference for thedisclosure contained therein regarding the use of Angptl4 polypeptides).

1. Patients with Nephrotic Syndrome Have Increased Levels of CirculatingAngptl4

Patients with Nephrotic syndrome have increased circulating levels ofAngptl4 polypeptide. 200 μg human plasma from patients (n=4patients/group) with diagnosed with MCD and MN and patients in MCDrelapse were analyzed by 2D gel electrophoresis and Western blots wereprepared using anti-Angptl4 antibodies (FIG. 1A). FIG. 1A shows thatonly patients with MCD relapse and MN had increased levels of Angptl4(indicated by arrows). This form of Angptl4 exists as a neutral pI formand is present as monomers and oligomers.

2. aP2-Angptl4 TG Rats Have Increased Circulating Levels of Angptl4

A transgenic rat models for adipocyte specific Angptl4 overexpressionwas developed and is shown in FIG. 1B (aP2-Angptl4 TG). Analysis of mRNAexpression in organs that normally express Angptl4 confirmed specificityof expression, with Angplt4 being detected in brown adipose tissue (BAT)and white adipose tissue (WAT) (FIG. 1C).

2D gel electrophoresis of 200 μg plasma, followed by Western blottingusing an anti-Angptl4 antibody revealed that heterozygous aP2-Angptl4 TGrats had higher circulating Angptl4 levels than wild type rats (FIG. 1D)(age 3 months, n=3 blots/group). FIG. 1E shows 2D gel electrophoresis of200 μg plasma, followed by Western blotting using anti-Angptl4 andanti-V5 antibodies show the presence of adipose tissue secretedV5-tagged Angptl4 in the plasma of aP2-Angptl4 TG rats. 2D gelelectrophoresis of immunoprecipitated Angptl4 from aP2-Angptl4 TG ratplasma (using an antibody specific for the N-terminus of Angptl4),followed by Western blotting using anti-Angptl4 or anti-lectin. SNA Iantibodies revealed the presence of sialylated Angptl4 polypeptide inthe circulation.

The aP2-Angptl4 TG rats had morphologically normal glomeruli by light(FIG. 1G) and electron microscopy (not shown), and glomerular Angptl4expression was unchanged. This is in contrast to podocyte specificexpression of Angptl4, where such expression resulted in glomerulardefects, including progressive development of foot process effacementbetween age one to five months (see U.S. Provisional application No.61/351,865 (filed 5 Jun. 2010), which is hereby incorporated byreference for such teaching).

Immunogold EM using anti-V5 antibody to specifically detect transgeneexpressed protein in 3 month old heterozygous aP2-Angptl4 TG male ratsdemonstrated detection selectively on the endothelial surface,indicating that circulating Angptl4 middle and high order oligomers donot enter the GBM and have receptors on the endothelial surface. Theeffects of circulating Angptl4 is relevant to both human andexperimental nephrotic syndrome, since adipose tissue upregulation ofAngptl4 is noted in later stages of nephrotic syndrome, when proteinuriais on the decline.

3. Relationship of Increased Circulating Levels of Angptl4 withProteinuria and Albuminuria

To examine the relationship between circulating levels of Angptl4proteinuria, including albuminuria, proteinuria was analyzed inaP2-Angptl4 TG rats. FIG. 2A shows that that aP2-Angptl4 TG do notexhibit proteinuria as determined by analysis of urinary proteins. InFIG. 2A urinary proteins were analyzed by GelCode blue stained SDS PAGE(3 μg/lane, except MCD remission) (densitometry readings are providedunder each lane). The intact albumin band is observed at 70 kDa(indicated by arrow). As can be seen, WT rats, aP2-Angptl4 TG rats andMCD patients in remission showed little or no intact albumin in theanalysed urinary samples, wherein NPHS2-Angplt4 TG rats (a rattransgenic model having podocytes specific Angptl4 expression and shownto develop MCD with proteinuria; see U.S. Provisional application No.61/351,865 (filed 5 Jun. 2010), which is hereby incorporated byreference for such teaching), MCD relapse, MN relapse and PAN rats (arat model of nephrotic syndrome) showed strong albumin stainingindicative of albuminuria. FIG. 2B shows that female heterozygousaP2-Angptl4 female TG rats had decreased albuminuria as compared to WTlittermate controls. FIG. 2C shows the same results for aP2-Angptl4heterozygous male TG rats. FIG. 2D shows that aP2-Angptl4 TG ratsexhibited reduced proteinuria in the puromycin nephrosis (PAN model; arat model of nephrotic syndrome) as compared to WT littermates. Asdemonstrated above, aP2-Angptl4 TG rats have higher circulating Angptl4levels that migrate at or around neutral isoelectric point, and issialylated. These results show a role for circulating Angptl4 inreducing proteinuria and nephrotic syndrome.

Since endothelial binding of adipose tissue secreted Angptl4 bound toglomerular endothelium, experiments were conducted to determine theeffect of recombinant Angptl4 on glomerular epithelial cells (GEnCs) toinvestigate whether lower baseline albuminuria and less PAN inducedproteinuria in this rat model were mediated by glomerular endothelialprotection. GEnCs were subject to oxidative injury by addition ofhydrogen peroxide and into the culture media and incubated withconcentrated supernatant (600 μg/well) from the control stable cellline, Angptl4-HEK293 cell line (secreting high isoelectric point (pI),hyposialylated Angptl4) or Angptl4-HEK293 cell line incubated withManNAc (neutral pl, normally sialylated Angptl4). It should be notedthat the high pI form of Angptl4 is secreted in large amounts frompodocytes in MCD. Release of LDH was assessed as a marker of cellinjury, Control cells without hydrogen peroxide injury were given arelative score of 1. High pI Angptl4 increased GEnC injury, whereasneutral pI Angptl4 (which comprises most of circulating Angptl4) wassignificantly protective at all measured time points. (n=3readings/condition).

Upregulation of Angptl4 in wild type rats on PAN Day 6 was exclusivelyglomerular, whereas upregulation in adipose tissue was noted on Day 10when proteinuria and glomerular Angptl4 expression are on the decline(n=3 rats/sample) (FIG. 2F). Therefore, increases in circulating Angptl4levels are coincident with the protective effect of circulating Angptl4in nephrotic syndrome and reduction of proteinuria. The effects ofcirculating Angptl4 are likely to be relevant to both human andexperimental MCD, since adipose tissue upregulation of Angptl4 is notedin later stages of PAN when proteinuria is on the decline. Furthermore,increased circulating Angptl4 levels at baseline and after induction ofPAN in aP2-Angptl4 TG rats resulted in increased plasma triglyceridelevels (FIG. 2G) and reduced post-heparin lipoprotein lipase activity(FIG. 2H) as compared to wild type rat.

In order to demonstrate the effectiveness of the therapeutic delivery ofAngptl4 into the circulation, wild type Angptl4 or a control protein wasadministered to Buffalo/Mna rats, a model of FSGS, or to Wistar rats inwhich Thy1.1 nephritis, a short term model of mesangial injury, wasinduced (FIGS. 4A and B). Wild-type recombinant Angptl4 polypeptide wasgenerated by harvesting of recombinant protein. Angptl4-HEK293 stable orpcDNA3.1-HEK293 control stable cell lines were grown to confluence in 15cm dishes, washed twice with warm PBS, and incubated with serum freeDMEM without Phenol Red, with or without 25 mM ManNAc, for 48 hours.Cells were harvested and the supernatant concentrated. Concentratedsupernatant from one 15 cm dish was used at each injection time point.

Buffalo/Mna rats spontaneously develop lesions mimicking human FSGS ataround age 2 months, including focal and segmental lesions on lightmicroscopy, effacement of podocyte foot processes on electronmicroscopy, and proteinuria. The rats develop progressive increase inproteinuria as they age. The rats used in the above studies were maleand 5 months old. Anti-THY1.1 nephritis was induced by injection of 150μg of anti-THY1.1 (Ox-7 hybridoma) or control IgG IV into differentgroups of male Wistar rats (100-125 gm, n=4 rats/group).

In the Buffalo/Mna rat model, assessment of baseline proteinuria wasmade on Day 0. Angptl4 or control protein were injectedintra-peritoneally on two consecutive days (Days 1 & 2, arrows) intoBuffalo Mna rats (n=4 rats/group), Proteinuria was assessed on alternatedays, and expressed as a percentage of baseline values. Significantreduction in proteinuria was noted in recombinant Angptl4 treated rats.

In the THY1.1 nephritis model, proteinuria confirmed on Day 1. Rats wereinjected intravenously with either recombinant Angptl4 or controlprotein on two consecutive days (Days 1 & 2, arrows). Proteinuria wasthen assessed. As shown in FIG. 4B, proteinuria was lower in Angptl4treated rats throughout, and was statistically significant on Day 5.

These results show that therapeutic delivery of Angptl4 into thecirculation are an effective treatment for nephrotic syndrome, such asbut not limited to minimal change disease, focal segmentalglomerulosclerosis, membranous nephropathy/membranousglomerulonephritis, membranoproliferative glomerulonephritis or adiabetic condition, such as, but not limited to, diabetic nephropathy,diabetes mellitus, lupus nephritis or primary glomerular disease.Furthermore, these results show that therapeutic delivery of Angptl4into the circulation are an effective treatment for and conditionsrelated to nephrotic syndrome, such as but not limited to, proteinuria,hypercholesterolemia, hypertriglyceridemia and edema. In one embodiment,the Angptl4 polypeptide is a derivative with decreased LPL inhibitoryactivity, resistance to cleavage or a derivative described herein.Administration of such a derivative would retain the beneficial effectsof Angptl4 treatment without the negative effects associated withinhibition of LPL activity, such as increased plasma triglyceridelevels.

Methods for Examples 1-3 Cloning of Full Length Rat Angptl4, andGeneration of Antibody Against Full Length Recombinant Angptl4

The full length rat Angptl4 open reading frame of 1218 bp from ourprevious experiments (7), excluding the stop codon, was cloned intopcDNA3.1/V5-HisB for eukaryotic expression, and into pET28a forprokaryotic expression. The E. Coli expressed purified full lengthprotein was used to generate a polyclonal antibody in rabbits(Proteintech group, Inc. Chicago IL USA) that was tested by ELISA andWestern blot. Antibody reactive bands were excised from GelCode bluestained gels, trypsin digested and presence of Angptl4 peptide sequencesconfirmed by MALDI-TOF/TOF. Part of the antiserum was affinity purifiedto the antigen. Unless otherwise specified, all studies described usedthis antibody. An additional polyclonal antibody against the N-terminalpart of rat Angptl4 (amino acids 7-86 excluding signal peptide) wassimilarly raised in rabbits.

Induction of Proteinuria in Animal Models of Human Glomerular Disease

All animal studies were approved by the institutional IACUC. Inductionof animal models of proteinuria (n=4 rats/group) in WT rats aredescribed in previous publications in parenthesis: PAN (7), PHN (7), PANwith glucocorticoids (20), non-HIV collapsing glomerulopathy (18),nephrotoxic serum induced heterologous phase proteinuria (7).Anti-THY1.1 nephritis was induced by injection of 200 mcg of anti-THY1.1(Ox-7 hybridoma) or control IgG IV into different groups of male Wistarrats (100-125 gm, n=4 rats/group), and rats euthanized after 24 and 72hours.

The following techniques are described in prior publications: Taqmanreal time PCR (26), confocal imaging (7), in situ hybridization (27),immunogold EM (26), glomerular extraction and processing for Westernblot (26), assessment of charge by PEI method (28). For alcian bluestaining, the pH of the staining solution was adjusted to 2.5 usingacetic acid, and 0.1% nuclear fast red solution was used as acounterstain. Densitometry of glomerular basement membrane alcian bluestain (20 glomeruli/rat, 3 rats/group) was assessed using Image-Prosoftware (Media Cybernetics, Inc., Bethesda MD, USA). Densitometry of 2Dgel Western blots was assessed using Gel-Pro Analyzer software (MediaCybernetics, Inc.). Taqman real time PCR primers and probes are listedin FIG. 3 . For in situ hybridization, the digoxigenin labeled probe forrat Angptl4 included bp 1 to 548 of the ORF.

To obtain samples for post heparin LPL activity, rats were injectedintravenously with 10 units/100 gm weight of porcine heparin 15 minutesprior to euthanasia, and activity measured using an assay from RoarBiomedical, Inc (New York NY). Serum triglycerides were measured in thefasting state.

Injection of NTS into Angptl4−/− Mice

Angptl4−/− mice were provided to Sander Kersten as a kind gift from EliLily Corporation (Indianapolis IN USA). The study protocol was approvedby the Animal Studies Committee at Wageningen University. Eleven weekold male Angptl4−/− or +/+ mice (n=4 mice/group) were injectedintravenously with 1.5 mg γ2-NTS or normal sheep serum (Sigma AldrichSt, Louis MO USA), spot urine samples collected at 48 hours, miceeuthanized at 72 hours, plasma collected for biochemical measurements,and kidneys preserved for histological analysis. Urine albumin wasassessed by ELISA (Bethyl laboratories, Montgomery TX USA) and urinecreatinine measured by mass spectrometry. To assess for foot processeffacement, the mean width of foot processes was first measured incontrol treated Angptl4+/+ mouse transmission electron micrographs (10equally spaced readings/loop, 3 loops/glomerulus, 3 glomeruli/kidney, 3kidneys/group). Effacement was described as an over 2.5 fold increase inmean width. Total and effaced foot processes were counted in NTS treatedor control treated Angptl4−/− mice.

Studies with Archived Human Samples

Immunostaining of archived human kidney biopsies (n=5 biopsies percondition) was conducted on samples obtained via IRB approved protocolsat the Instituto Nacional de Cardiologia, Mexico City. Control kidneybiopsies used for these studies were sex and age matched protocolpre-transplant biopsies. Archival human sera for 2D gel electrophoresisand Western blot (n=4 samples/condition) were obtained from a previouslypublished study (29).

Generation of Transgenic Rats

aP2-Angptl4 TG rats (adipose tissue specific) construct was generated inthe vector that contained the 5.4 Kb mouse aP2 promoter construct (30)(purchased from Addgene Inc. Cambridge MA USA) by cloning the ratAngptl4 cDNA (including the signal sequence) with a C-terminal V5 tag atthe NotI site just upstream of the polyA tail.

Transgenic rats were generated by microinjection of the digested DNAconstructs into fertilized Sprague Dawley eggs (conducted at Universityof Michigan), implantation into pseudopregnant host Sprague Dawleyfemales, and the resulting offsprings were genotyped by routine PCR andTaqMan genomic DNA real time PCR strategy using construct specific andcontrol genomic prolactin primer and probe combinations (FIG. 3 ). Threefounder lines for adipose tissue specific expression were generated.Data from aP2-Angptl4 TG rat line 375 (3 copies), both stable over 4generations, are presented. Urinary total protein was assessed using theBradford method (Biorad laboratories, Hercules CA USA), and albuminuriaby ELISA (Bethyl laboratories, Montgomery TX USA).

In Vitro Studies with GEnCs

For GEnC studies, cultured rat GEnCs (32) were grown to 75% confluencein 6 well plates (n=3 wells/condition), washed twice with warm PBS,serum free RPMI containing 200 μM H2O2, along with 600 μg/well ofcontrol stable cell line supernatant, or Angptl4-HEK293 stable cell linesupernatant, or supernatant from ManNAc treated Angptl4-HEK293 cellline. Wells were sampled at 24, 36 and 48 hours. LDH release wasmeasured using the cytotoxicity detection kit (Roche Diagnostics,Mannheim Germany). OD 492 values were expressed as a ratio of readingsfrom wells in which no H₂O₂ or stable cell line supernatant was added.

Statistical Analysis

Analysis of difference in proteinuria or gene expression involving threeor more groups was conducted by ANOVA with post analysis testing usingGraphPad InStat software, Version 3.05. For comparison of two groups,the unpaired Students t test in Microsoft Excel 2003 was used.

4. Circulating Angiopoietin-like-4 Links Proteinuria withHypertriglyceridemia in Nephrotic Syndrome

A molecular basis for the relationship between proteinuria andhyperlipidemia (hypertriglyceridemia and hypercholesterolemia) innephrotic syndrome is not known. In this study, it is shown thatincreased plasma levels of the glycoprotein Angptl4 link proteinuriawith hypertriglyceridemia in nephrotic syndrome due to membranousnephropathy (MN), focal and segmental glomerulosclerosis (FSGS), andminimal change disease (MCD). Circulating Angptl4 had a near neutralisoelectric point (pI), and was mostly secreted from skeletal muscle,adipose tissue and heart after the establishment of moderate to severeproteinuria. In MCD, additional early podocyte expression of high pIAngptl4, that induces proteinuria, and neutral pI Angptl4 werepreviously shown. Using adipose tissue overexpressing Angptl4 transgenicrats (aP2-Angptl4) and recombinant Angptl4, it was shown thatcirculating Angptl4 reduced proteinuria by binding to glomerularendothelial αvβ5 integrin, while also inducing hypertriglyceridemia byblocking lipoprotein lipase (LPL) mediated triglyceride uptake.Hypertriglyceridemia was absent in nephrotic Angptl4−/− mice, NephroticAngptl4−/− and Itgb5−/− mice, and nephrotic rats injected with ananti-β5 integrin antibody had delayed recovery from peak proteinuria.Moreover, recombinant human Angptl4 with mutations at the LPL bindingsite could reduce proteinuria without affecting plasma triglyceridelevels. In summary, circulating Angptl4 reduces proteinuria while alsoinducing hypertriglyceridemia, and is mostly produced from peripheralorgans as a systemic response to nephrotic range proteinuria.

Background

Molecular pathways that link proteinuria with hyperlipidemia, two keyhallmarks of nephrotic syndrome, are not known. Hyperlipidemia has twocomponents, hypercholesterolemia and hypertriglyceridemial. In the past,hypercholesterolemia has been attributed to increased hepatic synthesisof lipoproteins in response to proteinuria and hypoalbuminemia (2).However, the precise molecular link between proteinuria and increasedhepatic lipoprotein synthesis remains unknown. The development ofhypertriglyceridemia has received much less attention. A majordeterminant of plasma triglyceride levels is the activity of endotheliumbound lipoprotein lipase (LPL), that regulates tissue uptake oftriglycerides from the circulation (3). Mice that lack LPL develop veryhigh triglyceride levels and die soon after birth (4). Prior studiesshow that the activity and expression of LPL protein, but not mRNA, arereduced in nephrotic syndrome (5). The molecular basis of this reductionin LPL protein activity and expression, or its relationship toproteinuria in nephrotic syndrome has not been determined.

A recent study from our laboratory showed increased expression ofAngptl4 in podocytes and in the circulation in minimal change disease(MCD) (6, 7). To study the biological role of podocyte-secreted Angptl4,two types of transgenic rat models were generated. NPHS2-Angptl4transgenic rats, that selectively overexpress Angptl4 within theglomerulus from podocytes, develop massive albuminuria withoutincreasing circulating Angptl4 levels. By contrast, aP2-Angptl4transgenic rats, that selectively overproduce and secrete Angptl4 fromadipose tissue, develop high circulating Angptl4 levels, but are notproteinuric. Further studies showed that podocytes secrete two distinctforms of Angptl4 in nephrotic syndrome: a high pI form that ishyposialylated, and neutral pI form that is sialylated. Treatment withthe sialic acid precursor N-acetyl-D-mannosamine (ManNAc) converts highpI Angptl4 to neutral pI Angptl4 in vivo, and significantly reducesalbuminuria/proteinuria. By contrast, circulating Angptl4 in normal andnephrotic rats and humans is comprised almost entirely of sialylatedneutral pI Angptl4.

Angptl4 is believed to block LPL activity (8) by inactivating LPL, whichreduces triglyceride update and results in hypertriglyceridemia (9).Population based sequencing studies of the human ANGPTL4 gene revealedlow plasma triglyceride levels in about 3% of the European-Americanpopulation that has an E40K variant (10). Subsequent studies showed thatrecombinant Angptl4 with the E40K variant is unable to inhibit LPLactivity in vitro (11). Angptl4 in circulation tends to cleave into anN-terminal fragment (contains LPL inhibiting region and a coiled coildomain, forms oligomers) and a C-terminal fragment (containsfibrinogen-like domain, remains monomeric), and mutating the Angptl4cleavage region between amino acids 161 and 164 improves the stabilityof the full length protein (11). We utilized these properties of Angptl4to develop mutants of potential therapeutic significance.

In the present study, the biological role of circulating Angptl4 innephrotic syndrome was investigated. We noted elevated levels of Angptl4and triglycerides, and reduced LPL activity in MN, FSGS and MCD. Inaddition, Angptl4−/− mice with nephrotic syndrome did not develophypertriglyceridemia. In rat models of nephrotic syndrome, elevatedcirculating Angptl4 originated primarily from skeletal muscle, adiposetissue and heart after severe proteinuria had developed. In experimentalMCD, some circulating Angptl4 also originated from podocytes. Elevatedcirculating Angptl4, whether by transgenic expression or injection ofrecombinant protein, increased triglyceride levels and reduced LPLactivity, but also reduced proteinuria in nephrotic rodents by bindingto glomerular endothelial αvβ5 integrin. Absence of β5 integrin, or itsin vivo blockage using specific antibodies, or absence of circulatingAngptl4, all slowed recovery from peak proteinuria. Angptl4 is thereforethe first direct molecular link between proteinuria andhypertriglyceridemia. It is likely that peripheral Angptl4 secretion isstimulated primarily to help reduce ongoing proteinuria in nephroticsyndrome, but also ends up binding to LPL and inducinghypertriglyceridemia.

Results Increased Circulating Angptl4 Levels Determine the Developmentof Hypertriglyceridemia in Nephrotic Syndrome

When compared with normal healthy volunteers, significantly elevatedfasting plasma Angptl4 levels were noted by ELISA in untreated patientswith nephrotic syndrome due to MCD, focal and segmentalglomerulosclerosis (FSGS), non-HIV collapsing glomerulopathy (CG), andmembranous nephropathy (MN) (FIG. 6(a), FIG. 12 ). To determine whetherelevated plasma Angptl4 levels can be correlated withhypertriglyceridemia and LPL activity in nephrotic syndrome, we studiedplasma Angptl4, triglycerides and post-heparin LPL activity in passiveHeymann nephritis (PHN, a model of MN) (12, 13), Buffalo Mna rats, thatspontaneously develop FSGS (14,15), and puromycin aminonucleosidenephrosis (PAN, a model of MCD)(12) (FIG. 6(b)). Fasting plasma Angptl4were elevated in these models after, but not before, they developedmoderate to severe proteinuria. In PHN and Buffalo Mna rats, significanthypertriglyceridemia was noted when plasma Angptl4 levels were elevatedand plasma LPL activity was reduced (FIG. 6(c)-(h)). In PAN,hypertriglyceridemia was present throughout proteinuria, persisted afterproteinuria had normalized, and correlated well with decline in LPLactivity (FIG. 6(i)-(k)). Overexpression of Angptl4 from adipose tissuein aP2-Angptl4 transgenic rats, that develop increased circulatingAngptl4 levels but no proteinurias, also induced hypertriglyceridemiaand reduced LPL activity (FIGS. 6(l) and (m)). By contrast, 3 month oldNPHS2-Angptl4 transgenic rats, in which Angptl4 overexpressed frompodocytes causes proteinuria but no leakage into the circulation, didnot develop elevated triglyceride levels or reduced LPL activity. Theseoverexpression studies suggest that entry of Angptl4 into thecirculation is required for the development of hypertriglyceridemia. Tostudy the relative importance of Angptl4 in the development ofhypertriglyceridemia in nephrotic syndrome, severe heterologous phasecomplement- and leukocyte-independent proteinuria was induced inAngptl4−/− and +/+ mice using γ2-nephrotoxic serum (NTS) (FIG. 6(n)).When compared with Angptl4+/+ mice, hypertriglyceridemia was absent inAngptl4−/− mice injected with NTS, despite these mice having significantproteinuria (FIG. 13(a)). These studies show that circulating Angptl4 isa critical mediator of hypertriglyceridemia in nephrotic syndrome.

Origin of Elevated Circulating Angptl4 in Nephrotic Syndrome

To determine the origin of increased circulating Angptl4, we conductedmulti-organ Angptl4 mRNA expression profiles in rat models of nephroticsyndrome. On PHN Day 9 (FIG. 7(a)), corresponding with elevatedcirculating Angptl4 levels (FIG. 6(b)) and heavy proteinuria (FIG.6(c)), prominent upregulation was noted in skeletal muscle, whiteadipose tissue (WAT), brown adipose tissue (BAT), and heart. Transientmild upregulation noted in glomeruli and liver earlier on Day 5 hadsubsided. In 4.5 month old Buffalo Mna rats (FIG. 7(b)) with elevatedAngptl4 levels (FIG. 6(b)) and moderate to severe proteinuria (FIG.6(f)), prominent upregulation was noted in the heart. Glomerularupregulation of Angptl4 is not seen in this model. In PAN, aself-limiting acute model of nephrotic syndrome, elevated circulatingAngptl4 levels occur throughout disease (FIG. 6(b)). Prominentupregulation of glomerular/podocyte Angptl4 during the crescendo phaseof proteinuria (Days 6 and 10) (FIG. 7(c)) was also previously described(6). This “glomerular” phase of Angptl4 upregulation (Day 6 and 10) wasfollowed by a “peripheral” phase (Days 14 and 21) during whichglomerular upregulation was absent, and prominent upregulation was notedin skeletal muscle, WAT and BAT. Since monogenic overexpression ofAngptl4 from podocytes in NPHS2-Angptl4 transgenic rats does notincrease circulating Angptl4 levels, 2-dimensional gel western blotstudies of rat plasma were performed during the “glomerular” phase aftermild PAN was induced in these rats (FIGS. 7(d), 7(e), 13(b)).Significantly higher circulating levels of Angptl4 were noted inNPHS2-Angptl4 transgenic PAN rats than wild type PAN rats. Thiscirculating protein was reactive with the anti-V5 antibody (FIG. 7(f)),thereby confirming its podocyte origin, since the transgene expressedprotein is V5-tagged at its C-terminal end. In keeping with increasedcirculating transgene overexpressed Angptl4, aP2-Angptl4 andNPHS2-Angptl4 transgenic rats with PAN had higher plasma triglyceridesand lower LPL activity than wild type PAN rats (FIGS. 7(g) and (h)).

Urinary Loss of LPL and Angptl4 in Nephrotic Syndrome

Unlike normal Sprague Dawley rats, nephrotic rats lost Angptl4 in theurine (FIG. 8(a)), thereby suggesting that circulating Angptl4 levelsunderestimate total Angptl4 production in nephrotic states. Whereasnormal rats degrade Angptl4 released LPL by hepatic uptake from thecirculation (16), additional loss was noted in the urine in nephroticsyndrome (FIG. 8(b), control serum blot in FIG. 13(c)). Reactivity with5D2 (FIG. 8(c), control serum blot in Supplementary FIG. 7(d)), amonoclonal antibody that selectively binds active LPL, suggests thatactive LPL is also lost in urine during heavy proteinuria. Also,nephrotic PAN rats were unable to increase LPL mRNA expression untilproteinuria was greatly diminished (FIG. 8(d)). By comparison,non-proteinuric aP2-Angptl4 transgenic rats could upregulate LPLexpression in skeletal muscle and heart in response to elevated Angptl4levels (FIG. 8(e)). These factors may contribute towards the maintenanceof Angptl4-mediated hypertriglyceridemia in nephrotic syndrome.

Increased Circulating Angptl4 Reduces Proteinuria

Induction of PAN in aP2-Angptl4 transgenic and wild type Sprague Dawleyrats revealed significantly lower proteinuria in transgenic rats (FIG.9(a)), suggesting that circulating Angptl4 has an anti-proteinuriceffect. To test whether this anti-proteinuric effect was specificallyinduced by circulating Angptl4, previously characterized rabbit anti-ratAngptl4 antibodies (6) were injected into wild type PAN rats after theonset of proteinuria to partially deplete circulating Angptl4 levels,and noted increased proteinuria (FIG. 9(b)). Next, recombinantsialylated rat Angptl4 was injected intravenously into Buffalo Mna rats(FIG. 9(c)), and rats with anti-THY1.1 nephritis, a model of mesangialinjury (FIG. 9(d)). In both cases, significant reduction of proteinuriawas noted.

Human Angptl4 Mutants With Reduced LPL Inactivation Reduce Proteinuria

In order to dissociate the LPL mediated effects of Angptl4 ontriglyceride uptake from its effects on proteinuria, pcDNA3.1 V5 His Bconstructs of human Angptl4 with mutations at two sites were generated(FIG. 10(a)). One set of mutations were made at or near a site known tobe important for binding to LPL (amino acids 40 or 39). Another set ofmutations were made in a region known to be involved in the cleavage offull length Angptl4 (amino acids 161 to 164).

Next, HEK 293 based stable cell lines were developed for these mutantand wild type plasmids, and recombinant protein containing supernatantharvested in serum free conditions. To ensure adequate sialylation ofAngptl4, ManNAc (N-acetyl-D-mannosamine) was added to the culture media.Wild type and recombinant Angptl4 were then assessed by Western blotusing anti-V5 antibody, and migration of the mutant proteins at theappropriate size and reduced cleavage were noted (FIG. 10(b)). Aftersingle intravenous injection of equal amounts of human recombinantAngptl4 into proteinuric Buffalo Mna rats, higher peak levels were notedfor the mutant proteins (FIG. 10(c)). Significant reduction ofproteinuria was noted for 2 weeks after a single injection in wild typeand mutant proteins (mean±SE of nadir as a percentage of baseline: wildtype 8525, 53.8±6.3; mutant 8501, 35.9±12.1: mutant 8515, 41.2±7.2)(FIG. 10(d)). Plasma triglyceride levels were significantly highercompared to baseline in wild type, but not in mutant, Angptl4 injectedrats at 3 and 6 hours after injection (FIG. 10(e)). Triglyceride levelswere significantly lower in mutant protein than in wild type proteininjected rats at 6 hours. Fasting triglyceride levels between 12 hoursand Day 17 were indistinguishable between wild type and mutant Angptl4injected rats, and were similar to controls (FIG. 13(e)).

Circulating Angptl4 Reduces Proteinuria by Binding to GlomerularEndothelial αvβ5 Integrin

Confocal imaging of aP2-Angptl4 TG rat kidney using anti-V5 antibodiesshowed that adipose tissue secreted Angptl4-V5 colocalized with theglomerular endothelium (FIG. 11(a)). Using immunogold EM, thisAngptl4-V5 was noted on the glomerular endothelial surface, mostly inthe region of the endothelial cell—glomerular basement membraneinterface (FIG. 11(b)), Using recombinant rat Angptl4 secreted fromstable cell lines, we showed that sialylated Angptl4 protein, whichmimics circulating Angptl4 in nephrotic states, protected cultured ratglomerular endothelial cells (GEnCs) from oxidative injury, whereashyposialylated Angptl4, a pro-proteinuric form that comprises about halfof podocyte-secreted Angptl4 in PAN and NPHS2-Angpt14 TG rats, increasesthe effects of oxidative injury (FIG. 11(c)).

Since Angptl4 was recently shown to bind 135 integrin (17), it wasdetermined whether the protective effects of circulating Angptl4 onproteinuria were mediated via binding to αvβ5 integrin present inglomerular endothelium. This protein:protein interaction was confirmedusing recombinant rat Angptl4 and plates coated with purified human αvβ5integrin, and noted strong dose dependent binding (R² 0.996) (FIG.11(d)). Induction of nephrotic syndrome using I2-NTS in β5 integrinknockout (ltgb5−/−) and wild type (Itgb5+/+) mice revealed much higherlevels of proteinuria in the knockout mice during the recovery phase(Days 5 and 7) (FIG. 11(e)), which corresponds with the peripheral phaseof circulating Angptl4 production from skeletal muscle and adiposetissue in this model (Supplementary FIGS. 14(a) and (b)). This suggeststhe decline from peak proteinuria (beyond Day 3) was influenced by thepresence of circulating proteins like Angptl4 that exertanti-proteinuric effects by binding αvβ5 integrin. To block the αvβ5integrin—Angptl4 interaction, an antibody against the extracellular partof 135 integrin (anti-135 integrin antibodies) or preimmune serum wasinjected into wild type (Sprague Dawley) rats (FIG. 11(f), SupplementaryFIGS. 14(c) and (d)) and aP2-Angptl4 transgenic rats (FIG. 11(g)) duringrecovery from peak proteinuria (beyond Day 10, corresponds to peripheralphase of circulating Angptl4 production) in PAN. A significant delay inrecovery was noted in both models. Finally, nephrotic syndrome wasinduced in Angpt4+/+ and Angptl4−/− mice and noted a significant delayin recovery from peak proteinuria (Day 7) during the peripheral phase ofcirculating Angptl4 production, which is absent in Angptl4−/− mice (FIG.11(h)). The lower level of proteinuria in Angptl4−/− mice during theglomerular phase is consistent with our previously published descriptionof podocyte secreted hyposialylated Angptl4 (6) as being one of severalcauses of proteinuria in this model.

Additional Mutants

Four addition mutant proteins were studied: 8496, 8506, 8511, and 8520.Each has at least one amino acid substitution at positions 39, 40, or161-164 as shown in FIG. 17 . HEK 293 based stable cell lines weredeveloped and cultured as described above to express mutant protein. Allfour mutant proteins were tagged with V5, with anti-V5 antibody, thenassessed by Western blot using anti-V5 antibody, and migration of themutant proteins at the appropriate size and reduced cleavage were noted.Results show that the amount of cleaved protein is significantly reducedin the mutants compared to the wild type via Western blot (FIG. 18 ),After single intravenous injection of equal amounts of mutant 8511protein into proteinuric Buffalo Mna rats, higher peak levels were notedfor the mutant 8511 (FIG. 19 ). Red arrows indicate single time pointwhen recombinant protein was injected. Two mutant human Angptl4 proteins(15 μg) were injected into Zucker Diabetic Fatty rats (a model ofdiabetic nephropathy and diabetic kidney disease, n=4 rats/group), afterwhich increased circulating levels of the mutant proteins (FIG. 20 )were noted, along with reduction in proteinuria (FIG. 21 ), but withoutsignificant increase in plasma triglyceride levels (FIG. 22 ). *P<0.05,**P<0.01. #P<0.05. All * values are relative to baseline Day 0 values.

Discussion

Without wishing to be bound by any given hypothetical model, this studyshows that circulating Angptl4 is a key molecular mediator in nephroticsyndrome (FIG. 15 ). It describes, for the first time, how two keycomponents of this syndrome, proteinuria and hypertriglyceridemia, arelinked at a molecular level. The glomerulus is central to thedevelopment of proteinuria by mechanisms that vary among differentdiseases, Using animal models of MN and FSGS, two conditions commonlyassociated with sub-acute onset of proteinuria and edema, it wasdemonstrated that peripheral organs, especially skeletal muscle, adiposetissue and heart, respond to increasing proteinuria by upregulatingAngptl4 expression. Circulating Angptl4, derived at this stageexclusively from these peripheral organs, has two potent effects. First,it binds to αvβ5 integrin in the glomerular endothelium and reducesproteinuria, thereby suggesting that the primary purpose of increasingcirculating Angptl4 is an attempt to reduce proteinuria. Second, andperhaps an unintended consequence of increasing circulating Angptl4levels, is its binding to LPL (its physiological function), reducedtriglyceride uptake, and hypertriglyceridemia. It is important toclarify that the link between proteinuria and hypertriglyceridemiadiscussed here does not relate to the primary pathogenesis ofproteinuria, but to its reduction/modification by circulating Angptl4.The lag between the onset of proteinuria and the development ofhypertriglyceridemia in human nephrotic syndrome is also explained byperipheral production of Angptl4, since it requires moderate to severeproteinuria (at least 3.5 grams/24 hours in humans) to develop thisresponse. Early, mild proteinuria in these two animal models was notassociated with a peripheral organ Angptl4 response, elevated plasmaAngptl4 or increased triglyceride levels. Since increased Angptl4secretion from peripheral tissues is also a physiological response tofasting (18), it is possible that increased peripheral production ofAngptl4 is part of a fasting-like response being used by the body tocurb excessive urinary protein loss in nephrotic syndrome.

Additional interesting lessons are learnt from PAN rats, a model of MCD,in which onset of edema and proteinuria is acute. Prior studies (6) showthat podocytes in MCD produce two distinct forms of Angptl4: a high pl,hyposialylated form that induces proteinuria, and a neutral pIsialylated form identical to circulating Angptl4. Among other factors,inadequate sialic acid substrate plays a major role in the production ofhigh pl Angptl4 by podocytes in MCD, and conversion of high pI Angptl4to neutral pI Angptl4 in vivo using the sialic acid precursorN-acetyl-D-mannosamine reduces proteinuria. Unlike FSGS and MN,glomerular upregulation of hyposialylated Angptl4 plays a key role inthe development of proteinuria in this disease. Circulating sialylatedAngptl4 remains elevated throughout the duration of the PAN model, withthe source being the glomerulus in the initial part (i.e. glomerularphase), and skeletal muscle and adipose tissue (i.e. peripheral phase)in the later stages. An in vitro study in this paper show that high pIAngptl4 increases, and neutral pl Angptl4 reduces endothelial injury inthe setting of oxidative stress. Further studies on high pI Angptl4 arebeyond the scope of this paper. All recombinant Angptl4 used for in vivostudies in this paper was the neutral pl sialylated form. As with ratmodels of MN and FSGS, PAN rats also develop a peripheral phase ofAngptl4 upregulation that contributes significantly to the decline ofproteinuria after Day 10.

A significant mediator to reduction in proteinuria in all models is theAngptl4—αvβ5 integrin interaction in glomerular endothelium, sinceabsence of β5 integrin or Angptl4 in knockout mice, or blockage of thisinteraction using antibodies directed against the extracellular part ofβ5 integrin reduces the rate of decline of proteinuria. Another effectof the peripheral phase of Angptl4 production in PAN is the persistenceof mild hypertriglyceridemia (Day 21) even after proteinuria hassubsided, Similar residual hypertriglyceridemia has been previouslydocumented in children with MCD after they go into remission (19). It ispossible that circulating Angptl4 may interact with other glomerularcell surface molecules as well to exert its protective effects. Therewere two reasons for pursuing binding of Angptl4 to glomerularendothelial αvβ5 integrin. First, confocal imaging and immunogoldelectron microscopy showed that Angptl4-V5 secreted from adipose tissuein aP2-Angptl4 transgenic rats binds specifically to endothelial cellsin the glomerulus. Second, αvβ5 is the only integrin expressed onglomerular endothelial cells in vivo shown to interact with Angptl4. Theother major glomerular endothelial integrin, αvβ3, does not interactwith Angptl4 (17) (confirmed by us, data not shown). The precisemechanism by which Angptl4 binding to endothelial αvβ5 integrin reducesproteinuria will be explored in the future. It is possible that putativeendothelial—podocyte feedback loops are affected.

Another interesting observation is that entry of Angptl4 into thecirculation after monogenic overexpression is organ dependent. Similarto a heart specific Angptl4 overexpressing transgenic mouse developed inthe past (20), monogenic over expression of Angptl4 in podocytes inNPHS2-Angptl4 rats does not automatically allow entry into thecirculation. By contrast, overexpression in adipose tissue (aP2-Angptl4transgenic rats (6), aP2-Angptl4 transgenic mice (18)) reliablyincreases circulating Angptl4 levels. The entry of podocyte secretedAngptl4 into the circulation, as noted in the Sprague Dawley rat PANglomerular phase and in NPHS2-Angptl4 transgenic rats with PAN, likelyrequires the activity of other as yet unidentified proteins produced inthe glomerulus. This also fits in well with human glomerular disease, inwhich expression of multiple genes and proteins is simultaneouslyaffected. Therefore, the systemic availability and effects ofcirculating Angptl4 is likely to be affected by other genes/proteinsaltered in multiple organs as part of the disease process, and also byurinary loss of Angptl4 and LPL in the nephrotic state.

The anti-proteinuric effects of circulating Angptl4 may already play apartial role in the efficacy of glucocorticoids used to treat manydifferent forms of glomerular disease. The effects of glucocorticoids onAngptl4 expression are organ dependent. Whereas they reduce Angptl4expression in podocytes in MCD (6), they have been shown to increaseadipose tissue expression of Angptl4 in mice (21). Future studies couldexplore whether glucocorticoids induce secretion of sufficient amountsof Angptl4 from adipose tissue into the circulation, whether this effectis dose dependent in vivo, and whether this also happens in nephroticsyndrome.

Other soluble proteins have been implicated in the pathogenesis of humanglomerular disease. Vascular endothelial growth factor, secreted frompodocytes and also present in the circulation, is shown to be involvedin the development of human thrombotic microangiopathy, and exerts itsbiological effects via specific receptors expressed on the endothelialand podocyte surface (22). Soluble fms-like tyrosine kinase 1 (23) andsoluble endoglin (24), secreted in excessive amounts from the placentain pre-ecclampsia, are involved in the pathogenesis ofglomeruloendotheliosis. These proteins, however, are implicated indisease pathogenesis and are not a systemic response to disease. Thesoluble urokinase receptor suPAR was recently shown to havepro-proteinuric effects exerted primarily by binding to podocyte αvβ3integrin (25). A common denominator between anti-proteinuric Angptl4 andpro-proteinuric suPAR is the interaction of both circulating proteinswith glomerular integrins. A follow up study by the same group showsthat suPAR levels are also increased in FSGS patients with mutations inthe NPHS2 gene (26). This would suggest that in these patients withNPHS2 mutations, the elevation of suPAR is a systemic response toglomerular disease. In such cases, SuPAR joins a class of circulatingproteins exemplified by Angptl4 that are increased in response toglomerular injury or proteinuria, and have potent effects that influencethe course of the underlying glomerular disease. This list will grow inthe near future once putative circulating proteins that influence thepathogenesis of non-HIV collapsing glomerulopathy (27, 28) areidentified.

Lastly, mutant forms of human Angptl4 are able to reduce proteinuriavery significantly (mean peak reduction around 60%) withoutsignificantly affecting plasma triglyceride levels, and are effectivefor at least two weeks after a single intravenous injection. Theserecombinant proteins hold promise for further development as therapeuticagents for human glomerular disease. In summary, circulating Angptl4 isan important biological mediator of nephrotic syndrome, and represents acritical link between proteinuria and hypertriglyceridemia.

Methods for Working Example 4 ELISA for Human and Rodent Angptl4

A sandwich ELISA to measure human Angptl4 from patient and controlplasma samples was purchased from R&D Systems (Minneapolis Min, USA).The standard curve was calibrated between 1.25 ng/ml and 40 ng/ml, andhad a R² value of 0.98. (Supplementary FIG. 16(a))

To measure rat and mouse Angptl4 in plasma, a new ELISA assay wasdeveloped. A sheep anti-rat Angptl4 antibody (5006B) was raised againstamino acids 22 to 101, and characterized for specificity by Western blotusing a previously published rabbit anti-rat Angptl4 antibody (6) aspositive control. Activity was also absorbed out using recombinantAngptl4 and loss of reactivity by Western blot and immunofluorescencewas documented. The assay was standardized using concentratedsupernatant from a previously published HEK293 based stable cell linethat secretes recombinant rat Angptl4. Wells were coated with between0.1 and 0.5 mg of concentrated supernatant. After blocking and washes,10 μg of sheep anti-rat Angptl4 antibody was added, followed by washes,16 ng/well of donkey anti sheep Ig HRP (Jackson laboratories), washes,and TMB system reagents, and measurement at OD 450 nm on an ELISA platereader. A standard curve with a linear relationship with a R² of 0.998was obtained (FIG. 16(b)). For analysis of rodent plasma, wells werecoated with 50 μl of plasma in duplicate, followed by steps as detailedabove. Readings from blank control wells, that contained all reagentsminus the study sample, were obtained and subtracted from readings ofthe study samples. A minimum of 4 samples were measured for each timepoint.

Human plasma samples used for ELISA assay were obtained from IRBapproved studies conducted at UAB (PI Chugh), Instituto Nacional DeCardiologia, Mexico City (PI Avila-Casado), and from previouslypublished studies (6).

Animal Studies

The generation and characterization of NPHS2-Angptl4 and aP2-Angptl4transgenic rats was previously published (6). Buffalo Mna rats wereobtained via MTA from Dr. Masao Mitsuyama at Kyoto University, KyotoJapan. Unless otherwise stated, all comparisons for Buffalo Mna ratswere made with age and sex matched Sprague Dawley and Wistar rats. Sinceresults were similar, only data from comparisons with Sprague Dawleyrats is presented. Itgb5−/− and control 129S1/SvlmJ mice were purchasedfrom Jackson Laboratories (Bar Harbor ME USA). Angptl4−/− mice wereprovided to Sander Kersten by Eli Lilly Corporation. All studies withAngptl4−/− mice were approved by the Animal Ethics Committee atWageningen University. All other animal studies were approved by theInstitutional Animal Care and Use Committee at the University of Alabamaat Birmingham.

Induction of single intravenous dose PAN (n=4 rats/group), PHN (n=4rats/group) and anti-THY1.1 nephritis (n=4 rats/group) was previouslydescribed (12, 13). For full dose PAN, puromycin aminonucleoside (SigmaChemical Company, St. Louis MO USA) 15 mg/100 gram was used. For mildPAN, dose was reduced to 7.5 mg/100 grams. Induction of complement- andleukocyte-independent nephrotic syndrome (n=4 mice/group) using the γ2fraction of sheep anti-rat nephrotoxic serum (NTS, kind gift from DavidSalant, Boston Medical Center) was previously described (29). For animalstudies in which rabbit anti-rat Angptl4 antibody (6) as injected intoPAN rats (n=3 rats/group), 500 μl of antibody or preimmune serum wasinjected in each dose. Depletion of circulating Angptl4 by the antibodywas confirmed by western blot. The volume of anti-β5 integrin antibodiesor pre-immune serum injected per dose in rat PAN studies was as follows:Sprague Dawley rat PAN (250 μl); aP2-Angptl4 transgenic rat PAN (500μl).

For multiorgan gene expression studies, organ samples were snap frozenin liquid nitrogen immediately after euthanasia (3 rats or mice/organsample, pooled). White adipose tissue was obtained from the abdomen,brown adipose tissue from the interscapular area, skeletal muscle fromthe thigh, liver frozen intact or samples from both left and right lobe,heart frozen intact, and rat kidneys frozen and used subsequently forglomerular isolation. In mouse experiments, kidneys were perfusedthrough the heart immediately after euthanasia using dynabeads, and thenused for glomerular isolation. Twelve cDNA templates were generated fromeach pooled organ, and gene expression assessed by Taqman real time PCR.

Injection of Recombinant Rat and Human Angptl4

Harvesting of sialylated rat Angptl4 from a HEK293 cell based stablecell line was previously described (6). Concentrated HEK293 Angptl4 orempty pcDNA 3.1 V5 His vector stable cell line supernatant (1.8 mg,derived from approximately 200 ml of media) containing rat Angptl4 wasinjected per dose in the Buffalo Mna rat (n=4 rats/group) and the THY1.1rat (n=4 rats/group) studies. For studies in which recombinant humanwild type Angptl4, mutant Angptl4 and control protein were injected intoBuffalo Mna rats (n=3 rats/group), 55 μg of recombinant protein(quantified by ELISA) in concentrated supernatant, or equal amounts ofcontrol stable cell line supernatant (equalized by protein assay) wasused per dose.

Post Heparin LPL Activity

Rats were injected intravenously with 10 units/100 g weight of porcineheparin 15 minutes prior to euthanasia, and activity measured using anassay from Roar Biomedical, Inc (New York NY USA) (30). Serumtriglycerides were measured in the fasting state using an autoanalyzer(some studies) or a kit from Cayman Chemical Company (Ann Arbor MI USA).

The following techniques have been previously described: 18 hour urinecollection in metabolic cages, measurement of proteinuria, mouse urinealbumin and creatinine, 2D gel electrophoresis and Western blot,confocal imaging, immunogold electron microscopy, extraction of totalRNA, generation of cDNA templates (2 μg total RNA/template), real timePCR (6, 12, 13, 31). In real time PCR studies, a three-fold change ismRNA expression was taken as significant and has been validated by us inprior publications (13, 31). Western blot for LPL was conducted usinggoat anti-LPL antibody (Santa Cruz Biotechnology, Santa Cruz CA USA),and a 5D2 monoclonal antibody (gift from John Brunzell, University ofWashington) that specifically identifies active dimeric LPL (32).Densitometry of 2D gel Western blots was conducted using Image Quant TL7.0 software (GE Healthcare, Waukesha WI USA). Mouse anti-PECAM1antibody was purchased from BD Pharmingen (San Diego CA USA). Allsecondary antibodies used were purchased from Jackson ImmunoResearchlaboratories (West Grove PA USA), and had minimal background reactivityto non-target species.

Development of Human Angptl4 Mutant Constructs and Stable Cell Lines

A human Angptl4 clone was mutated using PCR based mutagenesis. Wild typeand mutant human Angptl4 clones in pcDNA 3.1 V5 His vector were used todevelop HEK 293 based stable cell lines as previously described (6).When used for harvesting protein, the serum free DMEM included 25 mMN-acetyl-D-mannosamine (ManNAc), a precursor of sialic acid, to ensureadequate sialylation of recombinant proteins secreted into the media.Proteins were harvested and supernatant concentrated as previouslydescribed (6). Recombinant human Angptl4 was quantified using ELISA.

Endothelial Cell Study

Cultured rat GEnCs grown in serum free conditions for 24 hours weresubjected to H₂O₂ (200 μM) induced stress, and co-incubated with equalamounts of concentrated supernatant from Angptl4-HEK293 orcontrol-HEK293 stable cell lines. LDH levels were measured at 24, 48,and 72 hours in the supernatant as a measure of cell injury using acytotoxicity kit (Roche Applied Science Indianapolis IN USA).

αvβ5 Integrin Plate Assay

96 well plates were coated with 5 ng human purified αvβ5 integrin/well,blocked with TBST with 1% BSA, followed by incubation with increasingamounts of concentrated supernatant from stable cell lines that secreterat Angptl4-V5. The V5 tag was detected using an anti-V5 HRP antibody(Life Technologies, Grand Island NY USA, 1: 2500), the reactiondeveloped using the TMB peroxidase substrate and solution (KPL, Inc.,Gaithersburg MD USA), and read on a Labsystems Multiscan MCC340 (ThermoFisher Scientific, Waltham MA USA) at 450 nm.

Generation and Characterization of Anti-β5 Integrin Antibodies

Fusion proteins were generated against parts of the extracellularsegment of human β5 integrin to generate two polyclonal antibodies inrabbits (antibody 8472A, amino acids 35-460, includes integrin betadomain; antibody 8472B, amino acids 461 to 719, includes integrin betatail). Both antibodies were tested for specificity by Western blotbefore and after absorbing out reactivity to recombinant human 135integrin. Pilot studies were conducted by inducing PAN in Sprague Dawleyrats (n=3 rats/group), and injecting two doses each antibodyintravenously during the recovery phase to assess for in vivo blockageof β5 integrin (i. e. slower recovery of proteinuria). Since theefficacy of 8472A was several times higher than 8472B, 8472A (FIG.16(c)) was used for further studies (n=4 rats/group). Injection of thisantibody intravenously, followed by confocal imaging of glomeruli,showed localization to the endothelium in glomeruli (FIG. 16(d)).

Statistical Analysis

Analysis of difference between two groups was conducted using theunpaired Students t-test in Microsoft Excel 2010. For three or moregroups, ANOVA with post analysis testing using GraphPad InStat software,Version 3.10 was used.

REFERENCES (EXCEPT FOR WORKING EXAMPLE 4)

-   1. Falk R, Jennette C, Nachman P H. Primary glomerular disease. In    The Kidney, Brenner B M, editor, 6^(th) edition, 1263-1349 (2000).-   2. Gutman, A. & Shafrir, E. Adipose tissue in experimental nephrotic    syndrome. Am. J. Physiol. 205, 702-706 (1963).-   3. Vaziri, N. D. Molecular mechanisms of lipid disorders in    nephrotic syndrome. Kidney Int. 63, 1964-1976 (2003).-   4. Shearer, G. C. & Kaysen G A. Endothelial bound lipoprotein lipase    (LpL) depletion in hypoalbuminemia results from decreased    endothelial binding, not decreased secretion. Kidney Int. 70,    647-653 (2006).-   5. Reaven, Kolterman, O. G. & Reaven, G. M. Ultrastructural and    physiological evidence for corticosteroid-induced alterations in    hepatic production of very low density lipoprotein particles. J.    Lipid Res. 15, 74-83 (1974).-   6. Tsukamoto, Y., Kokubo, T., Horii, A., Moriya, R. & Kobayashi, Y.    Lipoprotein derangement during steroid treatment in minimal-change    nephrotic syndrome. Nephron 73, 606-612 (1996).-   7. Liu, G., Clement, L., Kanwar, Y. S., Avila-Casado, C. &    Chugh, S. S. ZHX proteins regulate podocyte gene expression during    the development of nephrotic syndrome. J. Biol. Chem. 281,    39681-39692 (2006).-   8. Yoon, J. C. et al. Peroxisome proliferator-activated receptor    gamma target gene encoding a novel angiopoietin-related protein    associated with adipose differentiation. Mol. Cell. Biol. 20,    5343-5349 (2000).-   9. Kersten, S. et al. Characterization of the fasting-induced    adipose factor FIAF, a novel peroxisome proliferator-activated    receptor target gene. J. Biol. Chem. 275, 28488-28493 (2000).-   10. Kim, I. et al. Hepatic expression, synthesis and secretion of a    novel fibrinogen/angiopoietin-related protein that prevents    endothelial-cell apoptosis. Biochem. J. 346, 603-610 (2000).-   11. Yoshida, K., Shimizugawa, T., Ono, M. & Furukawa, H.    Angiopoietin-like protein 4 is a potent hyperlipidemia-inducing    factor in mice and inhibitor of lipoprotein lipase. J. Lipid Res.    43, 1770-1772 (2002).-   12. Ge, H., et al. Oligomerization and regulated proteolytic    processing of angiopoietin-like protein 4. J. Biol. Chem. 279,    2038-2045 (2004).-   13. Ge, H., Yanq, G., Yu, X., Pourbahrami, T. & Li, C.    Oligomerization state-dependent hyperlipidemic effect of    angiopoietin-like protein 4. J. Lipid Res. 45, 2071-2079 (2004).-   14. Romeo, S., et al. Population-based resequencing of ANGPTL4    uncovers variations that reduce triglycerides and increase HDL. Nat.    Genet. 39, 513-516 (2007),-   15. Romeo, S. et al. Rare loss-of-function mutations in ANGPTL    family members contribute to plasma triglyceride levels in    humans. J. Clin. Invest. 119:70-79 (2009).-   16. Eremina, V., et al. VEGF inhibition and renal thrombotic    microangiopathy. N. Engl. J. Med. 358, 1129-1136 (2008).-   17. Davis, B., et al. Podocyte-specific expression of angiopoietin-2    causes proteinuria and apoptosis of glomerular endothelia, J. Am.    Soc. Nephrol. 18, 2320-2329 (2007).-   18. Avila-Casado, C., et al. Proteinuria in rats induced by serum    from patients with collapsing glomerulopathy. Kidney Int. 66,    133-143 (2004).-   19. Mandard, S., et al. The fasting-induced adipose    factor/angiopoietin-like protein 4 is physically associated with    lipoproteins and governs plasma lipid levels and adiposity. J. Biol.    Chem. 281: 934-944 (2006).-   20. Clement, L., et al. Early changes in gene expression that    influence the course of primary glomerular disease. Kidney Int. 72,    337-347 (2007).-   21. Cazes, A, et al. Extracellular matrix-bound angiopoietin-like 4    inhibits endothelial cell adhesion, migration, and sprouting and    alters actin cytoskeleton. Circ. Res. 99, 1207-1215 (2006).-   22. Malicdan, M. C., Noguchi, S., Hayashi, Y. K., Nonaka, I. &    Nishino, I. Prophylactic treatment with sialic acid metabolites    precludes the development of the myopathic phenotype in the DMRV-h    IBM mouse model. Nat. Med. 15, 690-695 (2009).-   23. Galeano, B. et al. Mutation in the key enzyme of sialic acid    biosynthesis causes severe glomerular proteinuria and is rescued by    N-acetylmannosamine. J. Clin. Invest. 117, 1585-1594 (2007).-   24. Ruge, T. et al, Lipoprotein lipase in the kidney: activity    varies widely among animal species. Am. J. Physiol. Renal Physiol.    287, F1131-F1139 (2004).-   25. Koliwad, S. K. et al. Angiopoietin-like 4 (ANGPTL4/FIAF) is a    direct glucocorticoid receptor target and participates in    glucocorticoid-regulated triglyceride metabolism. J. Biol. Chem.    284, 25593-25601 (2009).-   26. Liu, G. at al. Neph1 and nephrin interaction in the slit    diaphragm is an important determinant of glomerular permeability. J.    Clin. Invest. 112, 209-221 (2003).-   27. Dijkman, H. B. P. M., Mentzel, S., de Jong, A. S. &    Assmann, K. J. M. RNA in situ hybridization using    digoxigenin-labeled cRNA probes. Biochemica 2, 23-27 (1995).-   28. Isogai, S., Mogami, K., Shiina, N. & Yoshino, G. Initial    ultrastructural changes in pore size and anionic sites of the    glomerular basement membrane in streptozotocin-induced diabetic rats    and their prevention by insulin treatment. Nephron. 83, 53-58    (1999).-   29. Bakker, W. W. et al. Altered activity of plasma hemopexin in    patients with minimal change disease in relapse. Pediatr. Nephrol.    20, 1410-1415 (2005).-   30. Graves, R. A., Tontonoz, P., Platt, K. A., Ross, S. R. &    Spiegelman, B. M. Identification of a fat cell enhancer: analysis of    requirements for adipose tissue-specific gene expression. J. Cell    Biochem. 49, 219-224 (1992).-   31. Yoshida, K., Ono, M., Koishi, R. & Furukawa, H. Characterization    of the 5′ regulatory region of the mouse angiopoietin-like    protein 4. Vet. Res. Commun. 28, 299-305 (2004).-   32. Zeng, L. et al. HMG CoA reductase inhibition modulates    VEGF-induced endothelial cell hyperpermeability by preventing RhoA    activation and myosin regulatory light chain phosphorylation.    FASEB J. 19, 1845-1847 (2005).-   33. Romeo, S. et al. Population-based resequencing of ANGPTL4    uncovers variations that reduce triglyceride and increase HDL.    Nature Genetics, 39, 513-517 (2007).-   34. Yin, Wu et al. Genetic variation in Angptl4 provides insight    into protein processing and function, J. Biol. Chem., 284,    13213-13222 (2009).

REFERENCES (WORKING EXAMPLE 4)

-   1. Nachman, P. H, Jennette, J. C. & Falk, R. Primary glomerular    disease. in The Kidney, 8^(th) edn. (ed. Brenner, B. M.) 987-1066    (Elsevier, Philadelphia, 2008).-   2. Marsh, J. B. & Drabkin, D. L. Experimental reconstruction of    metabolic pattern of lipid nephrosis: key role of hepatic protein    synthesis in hyperlipemia. Metabolism 9, 946-955 (1960).-   3. Vaziri N. D. Molecular mechanisms of lipid disorders in nephrotic    syndrome. Kidney Int. 63, 1964-1976 (2003).-   4. Weinstock, P. H. et al. Severe hypertriglyceridemia, reduced high    density lipoprotein, and neonatal death in lipoprotein lipase    knockout mice. Mild hypertriglyceridemia with impaired very low    density lipoprotein clearance in heterozygotes. J. Clin. Invest. 96,    2555-2568 (1995).-   5. Shearer, G. C. & Kaysen, G. A. Endothelial bound lipoprotein    lipase (LpL) depletion in hypoalbuminemia results from decreased    endothelial binding, not decreased secretion. Kidney Int, 70,    647-653 (2006)-   6. Clement, L. C. et al., Podocyte—secreted Angiopoietin-like-4    mediates proteinuria in glucocorticoid-sensitive nephrotic syndrome.    Nat Med. 17, 117-122 (2011).-   7. Chugh, S. S., Clement, L. C. & Macé, C. New insights into human    minimal change disease: Lessons from animal models. Am, J. Kid. Dis.    59, 284-292 (2012).-   8. Yoshida, K., Shimizugawa, T., Ono, M. & Furukawa, H.    Angiopoietin-like protein 4 is a potent hyperlipidemia-inducing    factor in mice and inhibitor of lipoprotein lipase. J. Lipid Res.    43, 1770-1772 (2002).-   9. Sukonina, V., Lookene, A., Olivecrona, T. & Olivecrona, G.    Angiopoietin-like protein 4 converts lipoprotein lipase to inactive    monomers and modulates lipase activity in adipose tissue. Proc.    Natl. Acad. Sci. USA 103, 17450-17455 (2006).-   10. Romeo, S. et al. Population-based resequencing of ANGPTL4    uncovers variations that reduce triglycerides and increase HDL. Nat.    Genet. 39, 513-516 (2007).-   11. Yin, W. et al. Genetic variation in ANGPTL4 provides insights    into protein processing and function. J. Biol. Chem, 284,    13213-13222 (2009).-   12. Clement L. et al. Early changes in gene expression that    influence the course of primary glomerular disease. Kidney Int, 72,    337-347 (2007).-   13. Liu, G., Clement, L., Kanwar, Y. S., Avila-Casado, C. &    Chugh, S. S. ZHX proteins regulate podocyte gene expression during    the development of nephrotic syndrome. J. Biol. Chem, 281,    39681-39692 (2006).-   14. Nakamura, T. et al. Sclerotic lesions in the glomeruli of    Buffalo/Mna rats. Nephron 43, 50-55 (1986).-   15. Le Berre, L. et al. Extrarenal effects on the pathogenesis and    relapse of idiopathic nephrotic syndrome in Buffalo/Mna rats. J.    Clin. Invest. 109, 491-498 (2002).-   16. Neuger, L. et al. Effects of heparin on the uptake of    lipoprotein lipase in rat liver. BMC Physiol. 4, 13 (2004).-   17. Zhu, P. et al. Angiopoietin-like 4 protein elevates the    prosurvival intracellular O2(−):H2O2 ratio and confers anoikis    resistance to tumors. Cancer Cell 19, 401-415 (2011).-   18. Mandard, S. et al. The fasting-induced adipose    factor/angiopoietin-like protein 4 is physically associated with    lipoproteins and governs plasma lipid levels and adiposity. J. Biol.    Chem. 281, 934-944 (2006).-   19. Zilleruelo, G., Hsia, S. L., Freundlich, M., Gorman, H. M. &    Strauss, J. Persistence of serum lipid abnormalities in children    with idiopathic nephrotic syndrome. J. Pediatr. 104, 61-64 (1984).-   20. Yu, X. et al. Inhibition of cardiac lipoprotein utilization by    transgenic overexpression of Angptl4 in the heart. Proc. Natl. Acad.    Sci. U.S.A. 102, 1767-1772 (2005).-   21. Koliwad, S. K. et al. Angiopoietin-like 4 (ANGPTL4,    fasting-induced adipose factor) is a direct glucocorticoid receptor    target and participates in glucocorticoid-regulated triglyceride    metabolism. J. Biol. Chem. 284, 25593-25601 (2009).-   22. Eremina, V. et al. VEGF inhibition and renal thrombotic    microangiopathy. N. Engl. J. Med. 358, 1129-1136 (2008).-   23. Maynard, S. E., et al. Excess placental soluble fms-like    tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction,    hypertension, and proteinuria in preeclampsia. J Clin. Invest. 111,    649-658 (2003).-   24. Venkatesha, S. et al. Soluble endoglin contributes to the    pathogenesis of preeclampsia. Nat. Med. 12, 642-649 (2006).-   25. Wei, C. et al. Circulating urokinase receptor as a cause of    focal segmental glomerulosclerosis. Nat. Med. 17, 952-960 (2011).-   26. Wei, C. et al. Circulating suPAR in Two Cohorts of Primary    FSGS. J. Am. Soc. Nephrol. 23, 2051-2059 (2012).-   27. Avila-Casado, C. et al. Proteinuria in rats induced by serum    from patients with collapsing glomerulopathy. Kidney Int, 66,    133-143 (2004).-   28. Chugh, S. S., & Clement, L. C. Telomerase at the center of    collapsing glomerulopathy. Nat. Med. 18, 26-27 (2012).-   29. Chugh, S. et al. Aminopeptidase A: A nephritogenic target    antigen of nephrotoxic serum. Kidney Int. 59, 601-613 (2001).-   30. Imamura, S. et al. A novel method for measuring human    lipoprotein lipase and hepatic lipase activities in postheparin    plasma. J. Lipid Res. 49, 1431-1437 (2008).-   31. Liu, G., et al. Neph1 and nephrin interaction in the slit    diaphragm is an important determinant of glomerular permeability. J.    Clin. Invest. 112, 209-221 (2003).-   32. Chang, S. F., Reich, B., Brunzell, J. D. & Will, H. Detailed    characterization of the binding site of the lipoprotein    lipase-specific monoclonal antibody 5D2. J. Lipid Res. 39, 2350-2359    (1998).

TABLE 4 LEGEND TO THE SEQUENCE LISTING SEQ ID NO: 1Variant 1 of human Angptl4 polypeptide SEQ ID NO: 2cDNA sequence of variant 1 of human Angptl4 SEQ ID NO: 3Variant 2 of human Angptl4 polypeptide SEQ ID NO: 4cDNA sequence of variant 2 of human Angptl4 SEQ ID NO: 5Rat Angptl4 polypeptide SEQ ID NO: 6cDNA sequence of rat Angptl4 polypeptide SEQ ID NO: 7Mouse Angptl4 polypeptide SEQ ID NO: 8cDNA sequence of mouse Angptl4 polypeptide SEQ ID NO: 9Variant 1 of human Angptl4 polypeptide with substitutions SEQ ID NO: 10Variant 2 of human Angptl4 polypeptide with substitutionsSEQ ID NOS: 11-22 PCR primers and probes (see text) SEQ ID NO: 23N-terminal multispecies consensus sequence with substitutionsSEQ ID NO: 24 Central multispecies consensus sequence with substitutionsSEQ ID NO: 25C-terminal multispecies consensus sequence with substitutionsSEQ ID NO: 26 N-terminal human consensus sequence with substitutionsSEQ ID NO: 27 C-terminal human consensus sequence with substitutionsSEQ ID NO: 28Mutant variant of human Angptl4 polypeptide DKMNVLAHGLLQLGQGLSEQ ID NOS: 29-90 Sequences from Table 2 SEQ ID NO: 91Reverse primer for amplification of aP2-Angptl4 construct SEQ ID NO: 92Probe for identification of aP2-Angptl4 construct SEQ ID NO: 93cDNA encoding peptide of SEQ ID NO: 80 SEQ ID NO: 94cDNA encoding the peptide of SEQ ID NO: 87

What is claimed:
 1. A mutant angiopoietin-like protein 4 (Angptl4)polypeptide having decreased LPL inhibitory activity, increasedresistance to cleavage, or a combination of the foregoing, wherein themutant Angptl4 polypeptide has at least 95% sequence identity with SEQID NO: 9 or SEQ ID NO: 10 and wherein positions 39 and 40 of SEQ ID NO:9 or SEQ ID NO: 10 are selected from the group consisting of: DK, DA, KEand AE, positions 76 and 80 of SEQ ID NO: 9 or SEQ ID NO: 10 areselected from the group consisting of: C, S, and A, and positions161-164 of SEQ ID NO: 9 or SEQ ID NO: 10 are selected from the groupconsisting of: GAAG (SEQ ID NO: 29), AAVV (SEQ ID NO: 69), GVVA (SEQ IDNO: 49), SGGG (SEQ ID NO: 87), and VAVA (SEQ ID NO: 90).
 2. The mutantAngptl4 polypeptide of claim 1, wherein positions 39 and 40 of SEQ IDNO: 9 or SEQ ID NO: 10 are DA and positions 161-164 of SEQ ID NO: 9 orSEQ ID NO: 10 are VAVA (SEQ ID NO: 90).
 3. The mutant Angptl4polypeptide of claim 2, wherein positions 76 and 80 of SEQ ID NO: 9 orSEQ ID NO: 10 are each C.
 4. The mutant Angptl4 polypeptide of claim 1,wherein positions 39 and 40 of SEQ ID NO: 9 or SEQ ID NO: 10 is DK andpositions 161-164 of SEQ ID NO: 9 or SEQ ID NO: 10 are selected from thegroup consisting of: GAAG (SEQ ID NO: 29), AAVV (SEQ ID NO: 69), GVVA(SEQ ID NO: 49), SGGG (SEQ ID NO: 87), and VAVA (SEQ ID NO: 90).
 5. Themutant Angptl4 polypeptide of claim 4, wherein positions 76 and 80 ofSEQ ID NO: 9 or SEQ ID NO: 10 are selected from the group consisting of:i) C, A, or 5, or ii) A and S.
 6. The mutant Angptl4 polypeptide ofclaim 1, wherein positions 39 and 40 of SEQ ID NO: 9 or SEQ ID NO: 10 isDA and positions 161-164 of SEQ ID NO: 9 or SEQ ID NO: 10 are selectedfrom the group consisting of: GAAG (SEQ ID NO: 29), AAVV (SEQ ID NO:69), GVVA (SEQ ID NO: 49), SGGG (SEQ ID NO: 87), and VAVA (SEQ ID NO:90).
 7. The mutant Angptl4 polypeptide of claim 6, wherein positions 76and 80 of SEQ ID NO: 9 or SEQ ID NO: 10 are selected from the groupconsisting of: i) C, A, or S, or ii) A and S.
 8. The mutant Angptl4polypeptide of claim 1, wherein positions 39 and 40 of SEQ ID NO: 9 orSEQ ID NO: 10 is KE and positions 161-164 of SEQ ID NO: 9 or SEQ ID NO:10 are selected from the group consisting of: GAAG (SEQ ID NO: 29), AAVV(SEQ ID NO: 69), GVVA (SEQ ID NO: 49), SGGG (SEQ ID NO: 87), and VAVA(SEQ ID NO: 90).
 9. The mutant Angptl4 polypeptide of claim 8, whereinpositions 76 and 80 of SEQ ID NO: 9 or SEQ ID NO: 10 are selected fromthe group consisting of: i) C, A, or S, or ii) A and S.
 10. The mutantAngptl4 polypeptide of claim 1, wherein positions 39 and 40 of SEQ IDNO: 9 or SEQ ID NO: 10 is AE and positions 161-164 of SEQ ID NO: 9 orSEQ ID NO: 10 are selected from the group consisting of: GAAG (SEQ IDNO: 29), AAVV (SEQ ID NO: 69), GVVA (SEQ ID NO: 49), SGGG (SEQ ID NO:87), and VAVA (SEQ ID NO: 90).
 11. The mutant Angptl4 polypeptide ofclaim 1, wherein: (a) positions 39 and 40 of SEQ ID NO: 9 or SEQ ID NO:10 is DK and positions 161-164 of SEQ ID NO: 9 or SEQ ID NO: 10 is GAAG(SEQ ID NO: 29), or AAVV (SEQ ID NO: 69); (b) positions 39 and 40 of SEQID NO: 9 or SEQ ID NO: 10 is DA and positions 161-164 of SEQ ID NO: 9 orSEQ ID NO: 10 is GVVA (SEQ ID NO: 49); (c) positions 39 and 40 of SEQ IDNO: 9 or SEQ ID NO: 10 is KE and positions 161-164 of SEQ ID NO: 9 orSEQ ID NO: 10 is SGGG (SEQ ID NO: 87); or (d) positions 39 and 40 of SEQID NO: 9 or SEQ ID NO: 10 is AE and positions 161-164 of SEQ ID NO: 9 orSEQ ID NO: 10 is VAVA (SEQ ID NO: 90).
 12. The mutant Angptl4polypeptide of claim 1, wherein the polypeptide is sialylated.
 13. Apharmaceutical composition comprising a therapeutically effective amountof the mutant Angptl4 polypeptide of claim 1 or claim 2 in combinationwith a pharmaceutically acceptable carrier.
 14. The pharmaceuticalcomposition of claim 13, wherein the therapeutically effective amountcomprises about 0.035-1100 mg of the mutant Angptl4 polypeptide.
 15. Thepharmaceutical composition of claim 13, formulated for intravenousdelivery or transdermal delivery.